Patent Application
20230203586
The present invention relates generally to the isolation and characterization of nucleic acid, particularly RNA, from small volume and self-collected samples, including fingerstick blood samples, swabs and saliva samples. The RNA derived is intact and of sufficient quality and quantity for RNA analysis, longitudinal RNA sequencing and global transcriptomic profiling.
RNA analysis, longitudinal RNA sequencing and global transcriptomic profiling are useful tools to identify and analyze biomarkers for disease, infection, exposure, susceptibility, drug response and toxicity (Frank M O et al (2019) BMC Medical Genetics 12:56; doi.org/10.1186/s12920-019-0500-0; Casamassimi A et al (2017) Int J Mol Sci 18(8):1652; Sheid A D et al (2018) J Immunol 200:1817-1928). In some disease and cancer studies, solid tissues or tumor samples are utilized, however, this is not practical in clinical studies or for continuous monitoring. Peripheral blood has advantages for biomarker evaluation and discovery due to its non-invasive collection and availability, particularly in comparison to solid tissue samples. A number of studies have demonstrated that transcriptomic changes in peripheral blood can serve as biomarkers of infection, exposure to xenobiotics, response to therapeutics or vaccines, or as indicators for pathological changes occurring in other tissues (Bushel P R et al (2007) Proc Natl Acad Sci 104(46):18211-6; Ramilo O et al (2007) Blood 109:2066-2077; Mejias A et al (2013) PLoSMed 10(11):e1001549; Hecker Met al (2013) Molec Neurobiol 48:737-756; Querec T D et al (2009) Nat Immunol 10:116-125).
The use of whole blood for transcriptomic profiling and RNA evaluation presents a number of significant technical challenges, including that RNA degradation and transcriptomic changes can occur quickly after the blood is drawn from subjects. Traditional reagents, such as citrate salts, heparin, and EDTA, inhibit blood clotting, but do not stabilize mRNA transcripts and altered gene regulation has been observed in whole blood samples, particularly when RNA is not immediately isolated (Debey S et al (2004) Pharmacogenomics 4:193-207; Rainen L et al (2002) Clin Chem 48:1883-1890). Moreover, the majority of RNA from whole blood encodes the globin protein, and sequencing that does not take this into consideration can yield results of low complexity (ie mostly globin mRNA, and few other unique or rare mRNA species).
Approaches for blood RNA stabilization have been developed to address the issues for RNA analysis from whole and peripheral blood samples (Asare A L et al (2008) BMC Genomics 9:474; Rainen L (2002) Clin Chem 48:1883-1890; Chai V et al (2005) J Clin Lab Anal 19_182-188). These include the PAXgene™ Blood RNA system (Qiagen) and the Tempus™ system (Applied Biosystems). In both systems, blood is immediately lysed when collected into the tube and RNA is stabilized using specific reagents. The PAX blood collection tube system and stabilization buffer is described including in U.S. Pat. Nos. 6,602,718 and 6,617,170. Aspects of the Tempus guanidinium-based stabilization agent are provided in U.S. Pat. No. 5,972,613. Both of these systems are designed for and require 2.5 mls or 3 mls of whole blood, which necessitates venipuncture, and are not suitable for small blood samples such as from a laboratory animal, an infant, or for any applicable means of self-collection.
Fingerstick blood collection is a practical and minimally invasive sample collection method that is used for a wide range of applications in routine clinical practice and can be implemented outside of clinical settings. For example, fingerstick sampling is used by millions of individuals to collect daily small blood volumes to monitor sugar or glucose levels. Finger stick blood collection would also be of value in subjects where it is commonly difficult to collect blood via venipuncture such as in infants and young children, elderly or ill individuals with compromised veins, intravenous drug addicts, and very obese individuals, in field studies in remote and under-developed areas, in military subjects or physically active athletes, or in other situations such as where a rapid sample is necessitated or applicable or in instances where collection of a large number of samples, including from many individuals, need to be obtained in a short amount of time.
The RNA collection and analysis systems currently in use and available, however, are not designed for small volume samples, such as finger stick blood samples or samples of one or a few droplets of blood. Collection of small volumes of blood via finger sticks is especially indicated for high frequency or repeated sample collection, such as to enable monitoring individuals in health and disease or infection. In addition, these systems are not applicable for alternative types of samples which may be time and sample volume critical such as naspharyngeal, nasal or throat swabs or aspirates. These are commonly utilized in direct and rapid patient assessment for virus infection, particularly respiratory virus infection, such as for influenza, so that infection can be quickly evaluated and treatment prescribed.
In order to implement and apply RNA isolation, evaluation and analysis more broadly and across various clinical and nonclinical scenarios and situations, there is a need for methods and a system to reliably and effectively sample and analyze RNA from small volume samples and alternative sample types that can be collected frequently, rapidly, in large number, in the field or at home by a relatively untrained individual or non-health professional or patient. There is a need for straightforward and dependable systems and methods whereby RNA can be isolated from small volume samples, self-collection samples, fingerstick samples and evaluated qualitatively and quantitatively with confidence and dependable results, particularly for whole transcriptome analysis and profiling.
The present invention general relates to methods for RNA isolation and RNA profiling and analysis of small volume samples and self-collected samples, wherein the RNA is of sufficient quality and quantity for whole transcriptome analysis and transcriptomic profiling. In embodiments of the method, a small volume sample may be from a patient or individual having a disease or infection or at risk for or suspected of disease or infection. In some embodiments, the patient or individual obtains or collects the small volume sample. In some embodiments, the patient or individual is assisted by a non-medical person in collection of the sample. In an embodiment, the sample is collected from a patient or individual by a non-medical person, such as a spouse, parent, friend, guardian, etc that is not medically trained or involved in any medical profession. Critically, the invention describes methods to obtain sufficient quality and quantity of RNA for a variety of analyses, ranging from quantitating individual RNA species to sequencing entire transcriptomes of high complexity.
In accordance with the method, small volume sample(s) is collected and combined with an RNA stabilization solution. In some embodiments, the RNA stabilization solution is capable of lysing the cells in the sample and of stabilizing RNA contained in the cells or cell lysate of the sample. In some embodiments, the RNA stabilization solution is capable of lysing the cells in the sample and of stabilizing RNA contained in the cells or cell lysate of the sample in a single step. In embodiments, the sample and RNA stabilization solution are mixed, vortexed or shaken when combined. In some embodiments, the sample may be stored or left at room temperature for up to a few or several hours prior to refrigeration. In some embodiments, the sample is then stored in refrigerated conditions, such as at about 40° F. or about 4° C. for a brief time. In some embodiments, the sample is then stored in refrigerated conditions, such as at about 40° F. or about 4° C. for a brief time, up to a day or a few or several days. In some embodiments, the sample may be stored or left at room temperature for up to a few or several hours, up to 2 hours, up to 3 hours, up to 3 or 4 hours, prior to refrigeration. In some embodiments, the sample is then stored in refrigerated conditions, such as at about 40° F. or about 4° C. for a brief time, up to a day or a few or several days. In some embodiments, the sample is stored in a freezer or in frozen temperature conditions, such as at about 30 or 32° F. or about 0° C., either after collection, after brief (2-4 hour) storage at room temperature, or after brief (1-2 day) refrigerated storage.
A small volume sample may be less than 500 μl, less than 300 μl, less than 250 μl, about 200-300 μl, less than 200 μl, about 100-300 μl, about 150-300 μl, about 100-250 μl, about 50-300 μl. In an embodiment, a small volume sample volume is about 100-300 μl.
In some embodiments, the sample may be the sample is a small volume blood sample, a sputum or saliva sample, or a nasal, nasopharyngeal or oropharyngeal swab, wash or aspirate. In embodiments, the small volume sample is a blood sample and is collected via fingerstick or heelprick. In an embodiment, the small volume sample is a blood sample and is collected via fingerstick. In embodiments, the fingerstick sample or heelprick sample may comprise blood droplets directly from a fingerstick or heelprick or a capillary tube may be utilized.
In some embodiments, the sample volume is less than 500 μl, less than 300 μl, less than 250 μl, about 200-300 μl, less than 200 μl, about 100-300 μl, about 150-300 μl, about 100-250 μl, about 50-300 μl. In some embodiments, the volume is less than 100 μl, less than 50 μl, about 10-50 μl, about 10-20 μl, about 10 μl, as small as 10 μl or less. In an embodiment, the sample volume is about 100-300 μl. In an embodiment, the sample volume is about 50-300 μl. In some embodiments, the sample volume is on the order of a blood droplet volume, or one or a few blood droplet volumes. In some embodiments, the blood or sample volume is that of a capillary tube volume, or less than a blood droplet volume. Capillary tube sample volumes may be on the order of 60-100 μl, 100-200 μl, 5-25 μl, 10-50 μl, less than 10 μl, 1-5 μl.
In some embodiments, the volume of RNA stabilization solution is less than 1 ml, about 500 μl or less, about 300 μl or less, about 200-300 μl, or about 250 μl, or about 200 μl. In an embodiment, the volume of RNA stabilization solution is about 300 μl or less, about 200-300 μl, or about 250 μl, or about 200 μl. In some embodiments, including wherein the sample volume is very low, such as on the order of less than 50 μl, or 10-50 μl, or about 10 μl, or less than 10 μl, the volume of RNA stabilization solution is appropriately low, such as on the order of less than 100 μl, less than 50 μl, less than 25 μl, as small as 10 μl or less.
In some embodiments, the sample is collected into a tube or wherein the tube or receptacle for receiving the small volume sample and containing RNA stabilization solution has a total volume capacity of 1.5 ml or less, 1.2 ml or less, or lml or less, or less than lml, or less than 500 μl, or less than 300 μl, or less than 200 μl. In an embodiment, the sample is collected into a tube or wherein the tube or receptacle for receiving the small volume sample and containing RNA stabilization solution has a total volume capacity of 1.5 ml or less, such as a microtainer tube. In an embodiment, the sample is collected in a tube which is suitable for small volumes, including very small volumes, such as a capillary tube. In an embodiment, the sample is collected into a capillary tube, which is suitable for small volumes, such as less than 100 μl, or even for very small volumes, such as less than 50 μl, less than 25 μl.
The invention provides a method for RNA profiling and analysis of small volume samples from a patient or individual comprising:
wherein the RNA is of sufficient quality and quantity for whole transcriptome analysis and transcriptomic profiling through RNA sequencing (RNAseq).
In embodiments of the method, the RNA is isolated using a process comprising:
In some embodiments, between steps (b) and (c), the resuspended precipitate containing the RNA or the aqueous phase containing the RNA is contacted with a solution or column to remove residual sample cell debris and/or to homogenize the sample cell lysate.
For embodiments using protease, the protease may be proteinase K. For embodiments using proteinase K, lysis buffers may also contain detergents, which both inactivate adventitious agents, lyse cells, and activate the proteinase K. Proteinase K has activity at 25° C., although it can be activated by putting the collected sample in hot tap water (typical tap water is set at a maximum of 120° F., which is about 48° C.; Proteinase K is optimally active at ˜55° C.).
In embodiments of the method, the RNA is isolated using a process comprising:
In embodiments of the method, the RNA is isolated using a process comprising:
In an embodiment, the RNA stabilization solution may be a mixture of chaotropic salt and phenol. In an embodiment, the chaotropic salt may be a guanidine salt or guanidine based. In an amboiment, the RNA stabilization soltion may be the PAXgene RNA stabilization solution.
In an embodiment, purification out of chaotropic salts such as guanidine, with detergent, can be used. The downstream purification as in step (c) may be precipitation, contact with a nucleic acid binding solid bead or semi-porous bead, such as a silica or carboxylated magnetic bead. Modification of lysis buffer for contact with silica or magnetic beads may be to include salt (e.g. sodium acetate) detergent (e.g. 0.2% sarkosyl), reducing agent (e.g. dithiothreotol, e.g. 75 mM). Purification may be accomplished using magnets to purify nucleic acids, by washing magnetic beads with bound nucleic acid in 75-80% ethanol or isorpropanol, twice, and then eluting RNA off the magnetic beads in pure RNase free double distilled water (ddH2O).
In some embodiments the sample is a small volume blood sample, a sputum or saliva sample, or a nasal, nasopharyngeal or oropharyngeal swab, wash or aspirate. In some embodiments, the sample is a small volume blood sample. In an embodiment, the small volume sample is a blood sample and is collected via fingerstick. In embodiments, the fingerstick sample may comprise blood droplets directly from a fingerstick or a capillary tube may be utilized.
In some embodiments of the method(s), the sample volume is less than 500 μl, less than 300 μl, less than 250 μl, about 200-300 μl, less than 200 μl, about 100-300 μl, about 150-300 μl, about 100-250 μl, about 50-300 μl. In an embodiment, the sample volume is about 100-300 μl. In some embodiments, the volume is less than 100 μl, less than 50 μl, about 10-50 μl, about 10-20 μl, about 10 μl, as small as 10 μl or less. In an embodiment, the sample volume is about 50-300 μl. In an embodiment, the sample volume is about 50-250 μl or is about 50-200 μl.
In some embodiments of the method(s), buffer and solution volumes are reduced to 20-40% or 20-30% of those utilized for isolation of RNA from a standard venipuncture blood sample.
In some embodiments, the RNA stabilization solution is a chaotropic salt such as guanidinium thiocyanate based or containing solution. In some embodiments chaotropic salts such as guanidinium thiocyanate based lysis buffers may also contain detergents, which synergize to inactivate adventitious agents, lyse cells. Detergents may include sarkosyl, SDS, or other ionic or non-ionic detergents. Kits/lysis solutions containing chaotropic salts such as guanidinium thiocyanate based lysis buffers with or without detergents, are stable, even up to for years. They can be shipped and used at room temperature. They are less toxic than household bleach, and can be mailed with adherence to suitable or such standards
In some embodiments, any buffers or solutions are made, prepared or generated with RNAse free water or buffers.
In embodiments of the method, any suitable and efficacious protease is utilized. Suitable proteases are known and available in the art. In embodiment, the protease is proteinase K. In some embodiments, the sample is contacted and treated with a protease at a temperature above room temperature. In embodiments, the sample and protease are heated for protease treatment. In an embodiment, the sample and protease are heated to 50-60° C. or incubated at a temperature of 50-60° C. In an embodiment, the sample and protease are heated to or incubated at 55° C.
In accordance with embodiments of the method, the method further comprises sequencing the RNA. RNA may be sequenced using any suitable or recognized method, steps, system(s) or kit(s), including manual, semi-automated or automated method(s), system(s) or kits. In some embodiments, kits such as Illumina TruSeq or Kapa Hyper Prep Kits are utilized.
In an embodiment, the isolated RNA is converted to cDNA. In some embodiments, the isolated RNA is converted to cDNA and may be cloned or a library prepared therefrom or containing or based on the cDNA(s).
In some embodiments, abundant RNA species or RNA species not of interest are removed prior to sequencing. In embodiments, globin mRNA, ribosomal RNA(s) or species specific RNAs are removed prior to sequencing. Methods, systems and kits for removal of globin RNA and/or ribosomal RNA are know and available to one skilled in the art. In some embodiments, systems or kits such as BlobinZero (Illumina), Ribo-Zero Gold, TruSeq Stranded total RNA library prep, Ribo-Zero Globin, GLOBINclear kit (THermo Fisher Scientific), QIAseqFastSelect RNA removal kit (Qiagen) may be utilized. In some embodiments, species specific probes may be utilize to select out certain RNAs.
In embodiments or the method, the patient or individual has a disease or infection or is at risk of or suspected of disease or infection.
In some embodiments, the method is for longitudinal screening by RNA profiling and analysis of small volume samples from one or more patient or individual, wherein the patient or individual has a disease or infection or is at risk of or suspected of disease or infection. In embodiments, small volume samples are collected in series or in regular or designated increments of hours, days, weeks or months. In embodiments, small volume blood samples are collected via fingerstick in series or in regular or designated increments of hours, days, weeks or months.
In some embodiments, small volume samples may be collected or additionally collected at outset of symptom(s), such as one or more symptom or recognized parameter indicative of or associated with a disease or infection. The disease may be an acute or chronic disease. The disease may be a relapsing and/or remitting disease. The infection may be a bacterial or viral infection. The infection may be with a known or unknown infectious agent. The infection may be with a known or unknown virus or bacteria.
In embodiments of the invention, systems and kits for use and application of the methods are provided.
In embodiments, a system or kit is provided for RNA profiling and analysis of small volume samples from a patient or individual comprising:
In embodiments, the system or kit may further comprise an envelope or mailing container for shipment of the sample to a laboratory or facility for RNA isolation and analysis.
In some embodiments, the system or kit may be for longitudinal RNA profiling and analysis of multiple small volume samples collected in series from a patient or individual over days, weeks or months comprising:
In some embodiments of the system or kit, the volume of RNA stabilization solution is less than lml, about 500 μl or less, about 300 μl or less, about 200-300 μl, or about 250 μl. In an embodiment, the volume of RNA stabilization solution is about 300 μl or less, about 200-300 μl, or about 250 μl. In some embodiments, including wherein the sample volume is very low, such as on the order of less than 50 μl, or 10-50 μl, or about 10 μl, or less than 10 μl, the volume of RNA stabilization solution is appropriately low, such as on the order of less than 100 μl, less than 50 μl, less than 2 μl, as small as 10 μl or less
In some embodiments of the system or kit, the tube or receptacle for receiving the small volume sample and containing RNA stabilization solution has a total volume capacity of 1.5 ml or less, 1.2 ml or less, or lml or less. In some embodiments, the tube or receptacle for receiving the small volume sample and containing RNA stabilization solution is a tube which is suitable for small volumes, including very small volumes, such as a capillary tube. In an embodiment, the tube or receptacle is a capillary tube, which is suitable for small volumes, such as less than 100 μl, or even for very small volumes, such as less than 50 μl, less than 25 μl.
Other objects and advantages will become apparent to those skilled in the art from a review of the ensuing detailed description, which proceeds with reference to the following illustrative drawings, and the attendant claims.
The patent or patent application contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the U.S. Patent and Trademark Office upon request and payment of the necessary fee.
In accordance with the present invention there may be employed conventional molecular biology, microbiology, and recombinant DNA techniques within the skill of the art. Such techniques are explained fully in the literature. See, e.g., Sambrook et al, “Molecular Cloning: A Laboratory Manual” (1989); “Current Protocols in Molecular Biology” Volumes I-III [Ausubel, R. M., ed. (1994)]; “Cell Biology: A Laboratory Handbook” Volumes I-III [J. E. Celis, ed. (1994))]; “Current Protocols in Immunology” Volumes I-III [Coligan, J. E., ed. (1994)]; “Oligonucleotide Synthesis” (M. J. Gait ed. 1984); “Nucleic Acid Hybridization” [B. D. Hames & S. J. Higgins eds. (1985)]; “Transcription And Translation” [B. D. Hames & S. J. Higgins, eds. (1984)]; “Animal Cell Culture” [R. I. Freshney, ed. (1986)]; “Immobilized Cells And Enzymes” [IRL Press, (1986)]; B. Perbal, “A Practical Guide To Molecular Cloning” (1984).
Therefore, if appearing herein, the following terms shall have the definitions set out below.
The term “rheumatoid arthritis” or “RA” refers to a chronic disease, which is immune-mediated and inflammatory and is an autoimmune disorder, affecting the lining of joints that causes joint pain, stiffness, swelling and decreased movement of the joints and can eventually result in bone erosion and joint deformity. RA is a systemic autoimmune disease characterized by the simultaneous inflammation of the synovium of multiple joints.
An “RA flare” or “flare” refers to a surge in immune-mediated and/or inflammatory activity that is periodically experienced by a patient(s) with RA. During a flare, the level of fatigue and joint symptoms such as pain, swelling, and stiffness temporarily increase. Flares are periods of increased disease activity during which people's arthritis symptoms, which typically include joint pain, swelling, and stiffness, are more severe. An RA flare can involve an exacerbation of any symptom of the disease, but most commonly includes intense stiffness in the joints. People with RA report these common symptoms of flares: increased stiffness in joints, pain throughout the entire body, increased difficulty doing everyday tasks, swelling, such as causing shoes not to fit, intense fatigue, flu-like symptoms.
As used herein, “RNA” is defined as at least two ribonucleotides covalently linked together. The RNA may be any type of RNA. Examples include mRNA, tRNA, rRNA, shRNA, circRNA, scaRNA, scRNA, snRNA, siRNA or Piwi-interacting RNA, or a pri-miRNA, pre-miRNA, miRNA, snoRNA, long ncRNAs, anti-miRNA, precursors and any variants thereof. Further examples of RNA include RNA of a virus, or RNA sequences derived from a virus genome. Even further examples include RNA of a bacteria. RNA may be single stranded or double stranded, or may contain portions of both double stranded and single stranded sequence. RNA may be synthesized as a single stranded molecule or expressed in a cell (in vitro or in vivo) using a synthetic gene. RNA may be obtained by chemical synthesis methods or by recombinant methods.
RNA may also encompass the complementary strand of a depicted single strand. Many variants of RNA may be used for the same purpose as a given RNA. Thus, RNA also encompasses substantially identical RNA and complements thereof. A single strand provides a probe that may hybridize to a target sequence under stringent hybridization conditions. Thus, RNA also encompasses a probe that hybridizes under stringent hybridization conditions.
As used herein, “pg” means picogram, “ng” means nanogram, “ug” or “μg” mean microgram, “mg” means milligram, “ul” or “μl” mean microliter, “ml” means milliliter, “l” means liter.
A “replicon” is any genetic element (e.g., plasmid, chromosome, virus) that functions as an autonomous unit of DNA replication in vivo; i.e., capable of replication under its own control.
A “vector” is a replicon, such as plasmid, phage or cosmid, to which another DNA segment may be attached so as to bring about the replication of the attached segment.
A “DNA molecule” refers to the polymeric form of deoxyribonucleotides (adenine, guanine, thymine, or cytosine) in its either single stranded form, or a double-stranded helix. This term refers only to the primary and secondary structure of the molecule, and does not limit it to any particular tertiary forms. Thus, this term includes double-stranded DNA found, inter alia, in linear DNA molecules (e.g., restriction fragments), viruses, plasmids, and chromosomes. In discussing the structure of particular double-stranded DNA molecules, sequences may be described herein according to the normal convention of giving only the sequence in the 5′ to 3′ direction along the nontranscribed strand of DNA (i.e., the strand having a sequence homologous to the mRNA). An “origin of replication” refers to those DNA sequences that participate in DNA synthesis.
A DNA “coding sequence” is a double-stranded DNA sequence which is transcribed and translated into a polypeptide in vivo when placed under the control of appropriate regulatory sequences. The boundaries of the coding sequence are determined by a start codon at the 5′ (amino) terminus and a translation stop codon at the 3′ (carboxyl) terminus. A coding sequence can include, but is not limited to, prokaryotic sequences, cDNA from eukaryotic mRNA, genomic DNA sequences from eukaryotic (e.g., mammalian) DNA, and synthetic DNA sequences. A polyadenylation signal and transcription termination sequence will usually be located 3′ to the coding sequence.
Transcriptional and translational control sequences are DNA regulatory sequences, such as promoters, enhancers, polyadenylation signals, terminators, and the like, that provide for the expression of a coding sequence in a host cell.
The term “oligonucleotide,” as used herein in referring to the probe of the present invention, is defined as a molecule comprised of two or more ribonucleotides, preferably more than three. Its exact size will depend upon many factors which, in turn, depend upon the ultimate function and use of the oligonucleotide.
The term “primer” as used herein refers to an oligonucleotide, produced synthetically, which is capable of acting as a point of initiation of synthesis when placed under conditions in which synthesis of a primer extension product, which is complementary to a nucleic acid strand, is induced, i.e., in the presence of nucleotides and an inducing agent such as a DNA polymerase and at a suitable temperature and pH. The primer may be single-stranded and must be sufficiently long to prime the synthesis of the desired extension product in the presence of the inducing agent. The exact length of the primer will depend upon many factors, including temperature, source of primer and use of the method. For example, for diagnostic applications, depending on the complexity of the target sequence, the oligonucleotide primer typically contains 15-25 or more nucleotides, although it may contain fewer nucleotides.
The primers herein are selected to be “substantially” complementary to different strands of a particular target DNA sequence. This means that the primers must be sufficiently complementary to hybridize with their respective strands. Therefore, the primer sequence need not reflect the exact sequence of the template. For example, a non-complementary nucleotide fragment may be attached to the 5′ end of the primer, with the remainder of the primer sequence being complementary to the strand. Alternatively, non-complementary bases or longer sequences can be interspersed into the primer, provided that the primer sequence has sufficient complementarity with the sequence of the strand to hybridize therewith and thereby form the template for the synthesis of the extension product.
A “protease” as defined herein is an enzyme that hydrolyses peptide bonds. Conventional proteases may be used. Proteinase K is an example. It is preferred that the specific activity of the protease be high to degrade proteins in what can be a protein-rich sample and thereby protect the RNA from ribonucleases. The specific activity as determined by the Chromozym assay of the protease in the mixture of biological sample and denaturing solution is for example at least about 0.1 U/ml, at least about 1 U/ml, at least about 2.5 U/ml, at least about 5 U/ml, or at least about 10 U/ml. In another embodiment, the specific activity of the protease in the mixture is between 0.1 and 1000 U/ml.
Reference throughout this specification to “one embodiment,” “an embodiment,” “one example,” or “an example” means that a particular feature, structure or characteristic described in connection with the embodiment or example is included in at least one embodiment of the present embodiments. Thus, appearances of the phrases “in one embodiment,” “in an embodiment,” “one example,” or “an example” in various places throughout this specification are not necessarily all referring to the same embodiment or example. Furthermore, the particular features, structures or characteristics may be combined in any suitable combinations and/or sub-combinations in one or more embodiments or examples. In addition, it is appreciated that the figures provided herewith are for explanation purposes to persons ordinarily skilled in the art and that the drawings are not necessarily drawn to scale.
As used herein, the terms “comprises,” “comprising,” “includes,” “including,” “has,” “having,” or any other variation thereof, are intended to cover a non-exclusive inclusion. For example, a process, article, or apparatus that comprises a list of elements is not necessarily limited to only those elements but may include other elements not expressly listed or inherent to such process, article, or apparatus.
Further, unless expressly stated to the contrary, “or” refers to an inclusive “or” and not to an exclusive “or”. For example, a condition A or B is satisfied by any one of the following: A is true (or present) and B is false (or not present), A is false (or not present) and B is true (or present), and both A and B are true (or present).
In accordance with the invention a method, kit and system have been established along with a clinical and technical protocol for isolation of RNA via repeated home blood collection of small volume blood samples using self-performed finger stick sampling by patients with a disease. The system enabled and allowed for longitudinal RNA sequencing (RNAseq). In an exemplary set of studies, rheumatoid arthritis (RA) patients were assessed and their RNA evaluated over a series of time points and correlated with clinical and physical parameters regarding RA flares. Samples were obtained from numerous (over 300) time points from eight flares over four years in an index patient, and over 200 time points from flares in three additional patients. A sampling method and RNA stabilization and isolation protocol were developed providing high quality intact RNA. Asssessments established that the RNAseq data from small volume blood finger stick samples correlated with blood cell counts from venipuncture blood draws. Transcripts were identified that were differentially expressed antecedent to RA flares. Transcriptomics of the patients prior to RA flares revealed a unique cell type, PRIME cells, in RA blood, which are predicted to become activated by B cells in the weeks prior to RA flare, and then migrate out of the blood to the synovium.
The methods and systems provided and enabled longitudinal genomic analysis via whole transcriptome analysis and total RNA sequencing (RNAseq). The studies provided herein establish that the collection system and RNA stabilization and isolation methods permits RNA sampling with valuable and consistent results. Among various applications for the system and methods, RNA profiling and longitudinal RNAseq analysis using the system and methods can reveal dynamic changes leading to flares of chronic inflammatory disease, provide indicators of clinical parameters and susceptibilities in disease or infection, reveal mechanisms via RNA activation and/or alteration in the progression of disease or infection or the susceptibility thereto, etc.
The present invention general relates to methods for RNA isolation and RNA profiling and analysis of small volume samples, wherein the RNA is of sufficient quality and quantity for whole transcriptome analysis and transcriptomic profiling.
Transcriptomics is the study of the ‘transcriptome,’ initially termed to signify an entire set of transcripts, and now widely understood to mean the complete set of all the ribonucleic acid (RNA) molecules expressed in some given entity, such as a cell, tissue, or organism. Transcriptomics can encompass everything relating to RNAs, including their transcription and expression levels, functions, locations, trafficking, and degradation. It can also include the structures of transcripts and their parent genes with regard to start sites, 5′ and 3′ end sequences, splicing patterns, and posttranscriptional modifications and covers all types of transcripts, including messenger RNAs (mRNAs), microRNAs(miRNAs), and different types of long noncoding RNAs (lncRNAs).
Modern transcriptomics often uses high-throughput methods to analyze the expression of multiple transcripts in different physiological or pathological conditions and this is rapidly expanding our understanding of the relationships between the transcriptome and the phenotype across a wide range of living entities. Whole-transcriptome analysis with total RNA sequencing (RNA-Seq) detects coding plus multiple forms of noncoding RNA and a goal of total RNA sequencing is to accurately measure gene and transcript abundance, and identify known and novel features of the transcriptome.
It is important to recognize that different levels of RNA evaluation and analysis require alternative amounts of RNA in terms or yield or quantity and in terms of quality. For instance, gene expression profiling experiments that are looking for a quick snapshot of highly expressed genes may only require a relatively small amount or lower quality RNA, particularly in as much as the amount of RNA from a highly expressed gene is more significant comparatively (as compared to a lower expressed or comparatively rare or small RNA) in a sample. Evaluation of targeted gene expression or assessing for the presence or absence of one or more targeted RNA may only require a relatively small amount or lower quality RNA, particularly in as much specific RNA probes or primer based isolation procedures may be utilized in the analysis. Experiments looking for a more global view of gene expression, and some information on alternative splicing, typically require a more mid level of quality and quantity of RNA. This encompasses most or many published RNA-Seq experiments for mRNA/whole transcriptome sequencing.
In important contrast, studies or experiments looking for or requiring an in-depth and full view of the transcriptome, to evaluate, identify or assemble new transcripts, to accurately measure gene and transcript abundance, or to identify known and novel features of the transcriptome require the highest quality and quantity of RNA available from samples. Thus, methods wherein RNA is isolated—even if from small or smaller volume samples—but is not quantitatively and qualitatively of the highest level, while suitable for some RNA analysis and study, will not be suitable for accurate and complete transcriptome profiling or longitudinal RNA profiling. There is an insufficient amount of all RNAs isolated to provide accurate and complete RNA information.
Total RNA-Seq analyzes both coding and multiple forms of noncoding RNA for a comprehensive view of the transcriptome and accurate and full results necessitate high quality RNA which is sufficient in quantity and yield to provide accurate, full length and comprehensive RNA sequences representing the full transcriptome. This then captures both known and novel features, allows researchers to identify biomarkers across the broadest range of transcripts, enables a more comprehensive understanding of phenotypes of interest and allows profiling of the whole transcriptome across a wide dynamic range.
The methods provided herein and in accordance with the invention provide RNA which is of sufficient quality and quantity for whole transcriptome analysis and transcriptomic profiling through RNA sequencing. Known and available methods for RNA isolation, if applied in a manner designed for larger volume samples, such as a standard venipuncture blood sample, or a sample of 2-3 mls of blood for example, do not result in RNA of suitable quality and quantity for whole transcriptome analysis and transcriptomic profiling through RNA sequencing when applied to small volume samples, particularly for example small blood samples from a fingerstick, or samples in the volume range of 100-300 μl blood.
In some embodiments, the sample may be a small volume blood sample, a sputum or saliva sample, or a nasal, nasopharyngeal or oropharyngeal swab, wash or aspirate. In embodiments, the small volume sample is a blood sample and is collected via fingerstick or heelprick. In an embodiment, the small volume sample is a blood sample and is collected via fingerstick. In embodiments, the fingerstick sample or heelprick sample may comprise blood droplets directly from a fingerstick or heelprick or a capillary tube may be utilized.
In some embodiments, the sample volume is less than 500 μl, less than 300 μl, less than 250 μl, about 200-300 μl, less than 200 μl, about 100-300 μl, about 150-300 μl, about 100-250 μl, about 50-300 μl, about 50-200 μl, about 50-150 μl. In an embodiment, the sample volume is about 100-300 μl. In some embodiments, the volume is less than 100 μl, less than 50 μl, about 10-50 μl, about 10-20 μl, about 10 μl, as small as 10 μl or less. In an embodiment, the sample volume is about 50-300 μl. In some embodiments, the sample volume is on the order of a blood droplet volume, or one or a few blood droplet volumes. In some embodiments, the blood or sample volume is that of a capillary tube volume, or less than a blood droplet volume. Capillary tube sample volumes may be on the order of 60-100 μl, 100-200 μl, 5-25 μl, 10-50 μl, less than 10 μl-5 μl. Capillary tubes on the order of these volumes are readily available commercially, such as from Sigma-Aldrich. The volume of choice or preference may be therein selected or as preferred.
A small volume sample may be from a patient or individual having a disease or infection or at risk for or suspected of disease or infection. In some embodiments, the patient or individual obtains or collects the small volume sample. In some embodiments, the patient or individual is assisted by a non-medical person in collection of the sample. In an embodiment, the sample is collected from a patient or individual by a non-medical person, such as a spouse, parent, friend, guardian, etc that is not medically trained or involved in any medical profession.
In accordance with the method, small volume sample(s) is collected and combined with an RNA stabilization solution. In some embodiments, the RNA stabilization solution is capable of lysing the cells in the sample and of stabilizing RNA contained in the cells or cell lysate of the sample. In some embodiments, the RNA stabilization solution is capable of lysing the cells in the sample and of stabilizing RNA contained in the cells or cell lysate of the sample in a single step. In embodiments, the sample and RNA stabilization solution are mixed, vortexed or shaken when combined. In some embodiments, the sample may be stored or left at room temperature for up to a few or several hours prior to refrigeration. In some embodiments, the sample is then stored in refrigerated conditions, such as at about 40° F. or about 4° C. for a brief time. In some embodiments, the sample is then stored in refrigerated conditions, such as at about 40° F. or about 4° C. for a brief time, up to a day or a few or several days. In some embodiments, the sample may be stored or left at room temperature for up to a few or several hours, up to 2 hours, up to 3 hours, up to 3 or 4 hours, prior to refrigeration. In some embodiments, the sample is then stored in refrigerated conditions, such as at about 40° F. or about 4° C. for a brief time, up to a day or a few or several days. In some embodiments, the sample is stored in a freezer or in frozen temperature conditions, such as at about 30 or 32° F. or about 0° C., either after collection, after brief (2-4 hour) storage at room temperature, or after brief (1-2 day) refrigerated storage.
In some embodiments, the volume of RNA stabilization solution is less than lml, about 500 μl or less, about 300 μl or less, about 200-300 μl, or about 250 μl. In an embodiment, the volume of RNA stabilization solution is about 300 μl or less, about 200-300 μl, or about 250 μl. In some embodiments, including wherein the sample volume is very low, such as on the order of less than 50 μl, or 10-50 μl, or about 10 μl, or less than 10 μl, the volume of RNA stabilization solution is appropriately low, such as on the order of less than 100 μl, less than 50 μl, less than 25 μl, as small as 10 μl or less
The RNA stabilization solution may be guanidinium based. The RNA stabilization solution may be a PAXgene based solution, a Tempus RNA based solution, a Trizol solution, a QIAzol-based solution, a Dxterity based solution system. Suitable guanidinium based solutions, such as guanidinium thiocyanate solutions are known. Guanidinium based solutions and methods have been previously described (for example Chomczynski P & Sacchi N. (1987) Anal. Biochem. 162: 156-159). Some solutions are or may be preferred and more advantageous or more suitable in the methods so as to generate RNA of sufficient quality and quantity for RNAseq and transcriptomic analysis or longitudinal analysis as provided herein.
The sample may collected into a tube or wherein the tube or receptacle for receiving the small volume sample and containing RNA stabilization solution has a total volume capacity of 1.5 ml or less, 1.2 ml or less, or lml or less, or 500 μl or less. In an embodiment, the sample is collected into a tube wherein the tube or receptacle for receiving the small volume sample and containing RNA stabilization solution has a total volume capacity of 1.5 ml or less, such as a microtainer tube. In some embodiments, the tube or receptacle for receiving the small volume sample and containing RNA stabilization solution is a tube which is suitable for small volumes, including very small volumes, such as a capillary tube. In an embodiment, the tube or receptacle is a capillary tube, which is suitable for small volumes, such as less than 100 μl, or even for very small volumes, such as less than 50 μl, less than 25 μl. Suitable sized tubes or containers are known and available in the art.
The invention provides a method for RNA profiling and analysis of small volume samples from a patient or individual comprising:
The RNA may be isolated using a process comprising:
In embodiments of the method, the RNA is isolated using a process comprising:
In embodiments of the method, the RNA is isolated using a process comprising:
In embodiments, all buffer and solution volumes are reduced to about 20-30%, 20-28%, about 25% of the volumes for standard venipuncture blood, which is on the order of a sample volume of 2.5 mls. Thus, while the sample volume is about 1/10th or 10% of the standard blood volume for commercial kits and methods, the buffers and solutions are reduced to about 20-30% or about 25%.
In commercial RNA isolation kits, such as the PAXgene Blood RNA kit the blood collection tube contains RNA stabilization solution appropriate for about 2.5 ml of sample volume. The PAXgene Blood RNA tube contains 6.9 ml of RNA stabilization solution, applicable for about 2.5 mls of blood. For the PAXgene Blood RNA tube, the relative ratio of sample volume to RNA stabilization buffer is about 0.36, or the stabilization solution volume is about 2.5-3 fold or about 2.76 fold the sample volume. In the present method, about 500 μl or less, about 300 μl or less, about 200-300 μl, or about 250 μl of RNA stabilization solution is present or provided for collection of the small volume sample. In the present method, about 500 μl or less, about 300 μl or less, about 200-300 μl, or about 250 μl of RNA stabilization solution is present or provided for collection of the small volume sample, wherein the sample volume is less than 500 μl, less than 300 μl, less than 250 μl, about 200-300 μl, about 250 μl, less than 200 μl, about 100-300 μl, about 150-300 μl, about 100-250 μl, about 50-300 μl, about 50-200 μl, about 50-150 μl. The range of sample volume to RNA stabilization buffer is on the order of about 5 fold to about 2 fold, about 5 fold to about 1 fold, about 3 fold to about 2 fold the sample volume. While the PAXgene kit blood collection tube contains 6.9 mls of RNA stabilization solution, in the instant methods the sample is combined with about about 250 μl or 0.25 mls which is a relative volume of 3-4%.
In commercial RNA isolation kits, such as the PAXgene Blood RNA kit, buffer volume for protease treatment is about 340 μl comprising 300 μl of buffer and 40 μl of protease. In the present method, buffer volume for protease treatment is about 74-75W comprising 65 μl of buffer and about 9 μl of protease. The relative volume percentage of the protease buffer and protease in the present method is about 20-22% or about 22%.
In some embodiments, between steps (b) and (c), the resuspended precipitate containing the RNA or the aqueous phase containing the RNA is contacted with a solution or column to remove residual sample cell debris and/or to homogenize the sample cell lysate.
The sample may be a small volume blood sample, a sputum or saliva sample, or a nasal, nasopharyngeal or oropharyngeal swab, wash or aspirate. In some embodiments, the sample is a small volume blood sample. In an embodiment, the small volume sample is a blood sample and is collected via fingerstick. In embodiments, the fingerstick sample may comprise blood droplets directly from a fingerstick or a capillary tube may be utilized.
In some embodiments of the method, the sample volume is less than 500 μl, less than 300 μl, less than 250 μl, about 200-300 μl, less than 200 μl, about 100-300 μl, about 150-300 μl, about 100-250 μl, about 50-300 μl. In an embodiment, the sample volume is about 100-300 μl. In some embodiments, the sample volume is less than 100 μl, less than 50 μl, less than 25 μl, 10 μl or less.
In some embodiments of the method, buffer and solution volumes are reduced to 20-40% or 20-30% or about 25% of those utilized for isolation of RNA from a standard venipuncture blood sample, such as a 2.5 ml or about 2.5 ml sample.
In some embodiments, the RNA stabilization solution is a guanidinium thiocyanate based or containing solution.
In some embodiments, any buffers or solutions are made or generated with RNAse free water or buffers.
In embodiments of the method, any suitable and efficacious protease is utilized. Suitable proteases are known and available in the art. In embodiment, the protease is proteinase K. In some embodiments, the sample is contacted and treated with a protease at a temperature above room temperature. In embodiments, the sample and protease are heated for protease treatment. In an embodiment, the sample and protease are heated to 50-60° C. or incubated at a temperature of 50-60° C. In an embodiment, the sample and protease are heated to or incubated at 55° C.
The purification/isolation method may be adapted for a may utilize a fully manual purification. In embodiments of manual purification centrifugation or a vacuum manifold, or a combination thereof, may be utilized, for example in order to pass solutions through columns. The purification/isolation method may be adapted for or may utilize Semi-automated purification. In embodiments of semi-automated purification, the lysis step and the precipitation or organic extraction step are carried out manually, while column purification is performed in an automated fashion, such as using an automated liquid handling system. Application of the isolation methods to fully automated purification is contemplated and an embodiment hereof, where all steps are performed using a fully automated system such as a fully equipped liquid handling system or a fully automated extraction system. Such fully automated systems are known and available in the art. In some embodiments, the fully automated systems are modified to adjust volumes, reagents, materials for small volume sample handling.
In embodiments of the method, commercial kits or RNA purification systems are modified. In embodiments, the PAXgene Blood RNA kit and process is modified for suitability and capability to provide for RNA isolation and RNA profiling and analysis of small volume samples, wherein the RNA is of sufficient quality and quantity for whole transcriptome analysis and transcriptomic profiling. In embodiments, the Tempus Blood RNA system and process is modified for suitability and capability to provide for RNA isolation and RNA profiling and analysis of small volume samples, wherein the RNA is of sufficient quality and quantity for whole transcriptome analysis and transcriptomic profiling.
The PAXgene protocol for Manual Purification of Total RNA from Human Whole Blood Collected into PAXgene Blood RNA Tubes is as follows (2015 Handbook):
Procedure
The PAXgene Blood RNA system and method is specifically and particularly designed and applicable for blood sample volumes of about 2.5 mls, which is on the order of 10 fold larger volumes than the methods herein are processing. The PAXgene Blood RNA system and handbook provide a Troubleshooting Guide for issues with the system and notes that this troubleshooting guide may be helpful in solving any problems that may arise. With regard to Low RNA yield, the Troubleshooting Guide indicates: “Less than 2.5 ml blood collected in PAXgene Blood RNA Tube. Ensure that 2.5 ml blood is collected in the PAXgene Blood RNA Tube” (see PAXgene Blood RNA Tube Product Circular). The PAXgene blood RNA system is admittedly not designed for or successfully applicable to small volume samples.
A comparison of the PAXgene Blood RNA system procedure and RNA isolation method with the methods provided herein including in Example 1, will demonstrate that the volumes utilized, particularly including in each of steps are significantly reduced and are approximately 20- % of the volume indicated. Sample volumes of approximately 1/10th or 10% volume size of those recommended and best for the Paxgene system can be processed with approximately 25% volume size of the buffers and solutions to successfully provide RNA of sufficient quality and quantity for whole transcriptome analysis and transcriptomic profiling through RNA sequencing.
In accordance with an embodiment of the method, in comparison with commercial RNA isolation kit volumes, such as particularly the PAXgene Blood RNA kit method and procedure outlines above, the volume of buffer (water) in step 2. is 1 ml, which is 25% of the 4 ml in the kit method. In accordance with an embodiment of the method, the volume of buffer in step 4. is 75 μl, which is 21.4% of the 350 μl in the kit method. In accordance with an embodiment of the method, the volume of buffer in step 5. is 65 μl buffer and 9 μl proteinase K, which is 21.7% of the 300 μl and 22.5% of the 40 μl in the kit method. In accordance with an embodiment of the method, the volume of ethanol solution in step 8. is 75μ1, which is 21.4% of the 350 μl in the kit method. In accordance with an embodiment of the method, the volume of buffer in step 11. is 100 μl, which is 28.5% of the 350 μl in the kit method. In accordance with an embodiment of the method, the volume of buffer in step 14. is 100 μl, which is 28.5% of the 350 μl in the kit method. Volume adjustments of buffers and solutions in the present method range from about 21% to about 29% or overall about 25%.
Robison and colleagues previously reported a general assessment of transcript profiling from fingerstick blood samples (Robison EH et al (2009) BMC Genomics 10:617, doi:10.1186/1471-2164-10-617). Only RNA quality and broad correlations of gene expression data using genechip analysis comparing fingerstick samples with whole blood samples were reported. Robison followed the PAXgene Blood RNA kit (product #762164) protocol for RNA isolation and purification, with the exception of one modification, wherein after the first spin, the pellet was washed with 1 mL RNase free water instead of 4 mL due to its small volume. Robison reported that they tested a scaled down version of the PAXgene protocol, but found that using the standard volumes of buffers and washes had no effect on the yields and were preferred as easier to employ. This is in sharp contrast to the studies and results reported and provided herein.
The methods herein may further comprise sequencing the RNA. RNA may be sequenced using any suitable or recognized method, steps, system(s) or kit(s), including manual, semi-automated or automated method(s), system(s) or kits. In some embodiments, kits such as Illumina TruSeq or Kapa Hyper Prep Kits are utilized.
As part of or commensurate with the methods herein, the isolated RNA may converted to cDNA. Methods for generating cDNA from RNA are well known and available to one skilled in the art. Any applicable and effective method should be suitable. The isolated RNA may be converted to cDNA for probing or specific primer applications, such as to assess expression or for sequencing of specific RNAs or gene products. The isolated RNA may be converted to cDNA for cloning purposes, to be inserted or prepared in a vector, for introducing into or preparing a library therefrom.
As part of or commensurate with the methods herein, the isolated RNA may amplified. In some embodiments, theRNA may be converted to cDNA and then amplified. Suitable methods and systems for amplification are known and available. For instance, methods, kits and systems for PCR amflication, including RT-PCR, wherein RNA is first reverse transcribed to cDNA and then amplifies are well known and available. Amplification methods and approaches may be useful particularly in the instances of small volume samples and/or where small amounts of RNA are being isolated. Another amplification approach, which is also useful for small volume or small quantity RNA samples, is loop-mediated isothermal amplification (LAMP). Combining LAMP with a reverse transcription step allows detection and evaluation of RNA. LAMP is carried out at a constant temperature (60-65° C.) and thus does not require a thermal cycler. LAMP mathods may utilize Bst (Bacillus stearothermophilus) DNA polymerase.
Abundant RNA species or RNA species not of interest may be removed prior to RNA sequencing. For example, globin mRNA, ribosomal RNA(s) and/or species specific RNAs may removed prior to sequencing. In some instances, globin RNA and ribosomal RNAs are both removed. This serves to eliminate highly prevalent RNAs or known RNAs which are not of interest from the isolated RNAs. Eliminating highly prevalent or irrelevant globin RNA or rRNAs may facilitate analysis of RNAs which are of interest or which are less prevalent and present in smaller amounts. Methods, systems and kits for removal of globin RNA and/or ribosomal RNA are know and available to one skilled in the art. In some embodiments, systems or kits such as BlobinZero (Illumina), Ribo-Zero Gold, TruSeq Stranded total RNA library prep, Ribo-Zero Globin, GLOBINclear kit (THermo Fisher Scientific), QIAseqFastSelect RNA removal kit (Qiagen) may be utilized. In some embodiments, species specific probes may be utilize to select out certain RNAs.
In embodiments or the method, the patient or individual has a disease or infection or is at risk of or suspected of disease or infection. The disease may be an acute or chronic disease. The disease may be a relapsing and/or remitting disease. The infection may be a bacterial or viral infection. The infection may be with a known or unknown virus or bacteria. A viral infection or virus may be an influenza virus, a coronavirus, an unidentified virus, an RNA virus. A bacteria may be a gram-positive bacteria. A bacteria may be a Streptococcus or Staphylococcus bacteria. A disease may be an inflammatory disease, an immune disease, an auto-immune disease, cancer.
In some embodiments, the method is for longitudinal screening by RNA profiling and analysis of small volume samples from one or more patient or individual, wherein the patient or individual has a disease or infection or is at risk of or suspected of disease or infection. In embodiments, small volume samples are collected in series or in regular or designated increments of hours, days, weeks or months. Small volume samples of a small volume blood sample, a sputum or saliva sample, or a nasal, nasopharyngeal or oropharyngeal swab, wash or aspirate may be collected. A combination of sample types or varying sample types may be collected. In embodiments, small volume blood samples are collected via fingerstick in series or in regular or designated increments of hours, days, weeks or months.
Samples may be collected in several hour increments, twice a day, three or four times a day, every 4-6 hours, daily, every morning, every evening, every morning and evening, once a week, one a month, every two months, every four months, every six months, several times a year. Samples may be collected to evaluate the effects of a drug or agent, for example prior to and/or following administration of a drug or agent. In some embodiments, small volume samples may be collected or additionally collected at outset of symptom(s), such as one or more symptom or recognized parameter indicative of or associated with a disease or infection. Samples may be collected prior to and after or upon the recognition or development of one or more symptom or disease or infection parameter. Samples may be collected upon the development of a fever, cough, pain or discomfort, rash, etc.
Systems and kits for use and application of the methods are provided. A system or kit is provided for RNA profiling and analysis of small volume samples from a patient or individual comprising:
In embodiments, the system or kit may further comprise an envelope or mailing container for shipment of the sample to a laboratory or facility for RNA isolation and analysis.
In an embodiment, with collection, the first drop of blood is removed, for example with a sterile gauze or cotton ball, so as to avoid tissue fluids that may produce inaccurate or less effective results. In an embodiment, the finger, heel etc, is cleansed with an alcohol or detergent solution, wipe or swab prior to collection, so as to remove any surface debris, loose cells or bacteria or dirt.
In some embodiments the lancet may be a small manual blade or may be a spring-loaded assembly or a self-contained disposable unit, such as wherein the blade is automatically retracted a holder after use. One such example is the Dynarex SensiLance pressure activated lancet.
In some embodiments, the system or kit may be for longitudinal RNA profiling and analysis of multiple small volume samples collected in series from a patient or individual over days, weeks or months comprising:
In some embodiments of the system or kit, the volume of RNA stabilization solution is less than 1 ml, about 500 μl or less, about 300 μl or less, about 200-300 μl, or about 250 μl.
In some embodiments of the system or kit, the tube or receptacle for receiving the small volume sample and containing RNA stabilization solution has a total volume capacity of 1.5 ml or less, 1.2 ml or less, or lml or less.
In the specification, numerous specific details are set forth in order to provide a thorough understanding of the present embodiments. It will be apparent, however, to one having ordinary skill in the art that the specific detail need not be employed to practice the present embodiments. In other instances, well-known materials or methods have not been described in detail in order to avoid obscuring the present embodiments.
Throughout this specification, quantities are defined by ranges, and by lower and upper boundaries of ranges. Each lower boundary can be combined with each upper boundary to define a range. The lower and upper boundaries should each be taken as a separate element.
Additionally, any examples or illustrations given herein are not to be regarded in any way as restrictions on, limits to, or express definitions of any term or terms with which they are utilized. Instead, these examples or illustrations are to be regarded as being described with respect to one particular embodiment and as being illustrative only. Those of ordinary skill in the art will appreciate that any term or terms with which these examples or illustrations are utilized will encompass other embodiments which may or may not be given therewith or elsewhere in the specification and all such embodiments are intended to be included within the scope of that term or terms. Language designating such non-limiting examples and illustrations includes, but is not limited to: “for example,” “for instance,” “e.g.,” and “in one embodiment.”
In this specification, groups of various parameters containing multiple members are described. Within a group of parameters, each member may be combined with any one or more of the other members to make additional sub-groups. For example, if the members of a group are a, b, c, d, and e, additional sub-groups specifically contemplated include any one, two, three, or four of the members, e.g., a and c; a, d, and e; b, c, d, and e; etc.
The invention may be better understood by reference to the following non-limiting Examples, which are provided as exemplary of the invention. The following examples are presented in order to more fully illustrate the preferred embodiments of the invention and should in no way be construed, however, as limiting the broad scope of the invention.
Rheumatoid arthritis (RA), like many inflammatory diseases, is characterized by episodes of quiescence and exacerbation (flares). The molecular events leading to flares are unknown. We established a method, kit and system along with a clinical and technical protocol for isolation of RNA via repeated home blood collection of small volume blood samples using self-performed finger stick sampling by RA patients. The system enabled and allowed for longitudinal RNA sequencing (RNAseq). Samples were obtained from 364 time points from eight flares over four years in our index patient, and 235 time points from flares in three additional patients. We developed a sampling method and RNA stabilization and isolation protocol providing high quality intact RNA. Asssessments established that the RNAseq data from small volume blood finger stick samples correlated with blood cell counts from venipuncture blood draws. We identified transcripts that were differentially expressed antecedent to flares and compared these to synovial single cell RNAseq (scRNAseq). Flow cytometry and sorted blood cell RNAseq in additional RA patients were used to validate the findings.
Consistent changes were observed in blood transcriptional profiles one to two weeks antecedent to RA flare. B cell activation was followed by expansion of a previously unexplored circulating CD45−/CD31−/PDPN+, PRe-Inflammatory MEsenchymal (“PRIME”) cell in RA patient blood, which shared features of inflammatory synovial fibroblasts. Circulating PRIME cells decreased during flares from all four patients, and flow cytometry and sorted cell RNAseq confirmed the presence of PRIME cells in 19 additional RA patients.
Longitudinal genomic analysis of RA flares reveals PRIME cells in RA blood, and suggests a model in which they become activated by B cells in the weeks prior to RA flare, and then migrate out of the blood to the synovium. These studies established that the collection system and RNA stabilization and isolation methods permits RNA sampling with valuable and consistent results. Among various applications for the system and methods, RNA profiling and longitudinal RNAseq analysis using the system and methods can reveal dynamic changes leading to flares of chronic inflammatory disease.
Introduction
Rheumatoid arthritis (RA) symptoms are highly dynamic, with stable periods interrupted by unpredictable flares of disease activity. Such waxing/waning clinical courses are characteristic of many autoimmune diseases, including multiple sclerosis (1), systemic lupus erythematosus (2), and inflammatory bowel disease (3,4), underscoring a need to develop approaches to understand what triggers transitions from quiescence to flare in autoimmune disease.
This study explores disease pathophysiology with a longitudinal, prospective analysis of blood transcriptional profiles in individual RA patients over time utilizing small volume blood sampling via patient at home finger stick collection. Previous microarray studies of RA blood samples from relatively sparse time series data have identified few significant gene changes associated with disease activity (5-8). Here we provide the first RA study to look for molecular changes in blood that anticipate clinical flares. To do so we developed and optimized methods by which RA patients themselves could collect high quality finger stick blood samples for RNA sequencing (RNAseq), facilitating weekly blood sampling for months to years.
We analyzed patient reports of clinical disease activity and RNAseq data from four patients across multiple clinical flares. In our most deeply studied index case, we assessed 364 time points by RAPID3 from eight flares over four years, and analyzed 84 time points assessed by RNAseq. Collecting samples longitudinally enabled a search for transcriptional signatures that preceded clinical symptoms. Comparing these blood RNA profiles to synovial single cell RNAseq (scRNAseq) data (9) provided evidence that a biologically coherent set of transcripts are significantly increased in the blood prior to symptom onset, and a subset of these decrease as the patients begin to experience symptoms. These latter transcripts overlap with and likely demarcate cellular precursors to a novel subset of synovial sublining fibroblast cell types detected in inflamed RA synovium using scRNAseq. Analysis in 19 additional RA patients corroborated our findings. Our data suggests a model in which a previously unexplored circulating mesenchymal cell type, detectable in the weeks prior to RA flare, becomes activated by B cells and subsequently leaves the blood, traffics to synovium, and contributes to disease activity. These studies were facilitated by the ability to isolate and analyze high quality RNA which validly represented changes in vivo from patient self-collected small volume blood samples (finger stick).
Methods
Patient Data
All patients met American College of Rheumatology/European League Against Rheumatism 2010 (10,11) criteria for RA and were seropositive for cyclized citrullinated protein antibody (CCP). Disease activity was assessed from home each week, or up to 4 times daily during escalation of flares, using the routine assessment of patient index data 3 (RAPID3) questionnaire (12). Disease activity was also assessed at clinic visits, each month, and during flares, using both the RAPID3 and the disease activity score 28 (DAS28), which incorporates tenderness and swelling from 28 joints, erythrocyte sedimentation rate (ESR) and patient global assessment of disease activity. Complete blood counts (CBC) including white blood cells (WBC), neutrophils, monocytes, lymphocytes, and platelets were performed by the clinical lab at Memorial Sloan Kettering Cancer Center. We collected 43 clinic visits from the index patient, and 25, 14 and 12 clinic visits for the other three patients studied longitudinally. Nineteen additional seropositive RA patients and 18 age and sex matched non-RA patients, for whom peripheral blood mononuclear cells (PBMC) were available, were also studied for the presence of PRIME cells by FACS and RNAseq analysis.
RNA Preparation from Finger Stick Blood
Patients self-performed finger sticks at home to collect three drops of blood into a microtainer tube prefilled with fixative (RNA stabilization solution), and samples were mailed overnight each week. RNA was extracted using the PAXgene RNA kit and purified per manufacturer's protocols, except the volume of all washes and elutions was decreased to about 25% of the recommended volume by the manufacturer. RNA was assessed using the Agilent BioAnalyzer for quantity and quality. For library preparation, we used the GlobinZero kit (EpiCentre #GZG1224) and Illumina's Truseq mRNA Stranded Library kit, with 11-12 PCR cycles for 5-8 nM input and sequenced on HiSeq2500 with 150 base paired-end reads. Reads were aligned to Gencodev18 using STAR and quantified using featureCounts (v1.5.0-p2). Samples with at least four million paired-end reads were retained for analysis.
A detailed protocol is provided below:
Should receive a box with patient questionnaire and finger stick sample
Place ice pack in cardboard box lined with chuck to thaw and dry
Make notes in Sample Log:
Finger Stick Sample
(adapted from PAXgene RNA handbook version 2, June 2015)
Before starting
Set temperature of shaker incubator to 55° C.
Warm Buffer BR2 (Binding Buffer) to 37° C. if there are precipitates (Binding Buffer contains a guanidine salt (guanidine thiocyanate) which can form highly reactive compounds when combined with bleach)
Prepare Buffer BR4 (Wash Buffer) by adding 4 volumes of 100% ethanol to obtain working solution
Prepare DNAse I stock by dissolving solid DNAse I (1500 Kunitz units; Qiagen, cat #79254) in 550 ul of RNAse free water and mix by inversion (1500 Kuntz Units/0.55 ml). Do not vortex, DNAse is sensitive to physical denaturation.
1. Remove PAXgene Blood RNA microtainer tube from freezer and allow to warm to room temperature (+/−1 hr)
2. Place PAX blood in 2 ml microfuge tube and centrifuge at 5000×g at room temperature for 10 minutes
3. Remove and discard supernatant. Add 1 ml RNAse-free water (from PAXgene kit) to wash the pellet.
4. Vortex to resuspend the pellet, then centrifuge for 10 minutes at 5000×g in a centrifuge. Remove and discard the supernatant.
5. Thoroughly resuspend the pellet in 75 ul of Buffer BR1 (from PAXgene kit) (Resuspension Buffer) by vortexing
6. Add 65 ul Buffer BR2 (from PAXgene kit) and 9 ul Proteinase K solution (from PAXgene kit).
7. Mix by vortexing and incubate for 10 minutes at 55° C. in shaking heat block (800 rpm).
8. Pipet lysate to lavender top PAXgene shredder spin column (which removes clumps) and spin for 3 minutes at 18,000×g.
9. Transfer supernatant of flow through (about 150 ul) ** be careful with this step since pellet is gooey and easily disrupted** to a new 1.5 ml microcentrifuge tube.
10. Add 75 ul of 100% ethanol. Mix by vortexing and centrifuge at 1000×g for 2 seconds to remove drops from inside the tube of lid. Do not centrifuge for longer than this or nucleic acids may pellet and reduce the RNA yield.
11. Apply 225 ul of sample to red top PAXgene RNA spin column sitting in a 2 ml processing tube (from PAXgene kit). Centrifuge at 8000×g for 1 minute. Place the PAXgene column in a new 2 ml processing tube and discard the old processing tube containing the flow through.
12. Pipet 100 ul Buffer BR3 (Wash Buffer) to the PAXgene column and centrifuge at 8000×g for 1 minute. Place the PAXgene column in a new 2 ml processing tube and discard the old processing tube containing flow through.
13. Pipet 5 ul DNAase I stock solution into 35 ul of Buffer RDD. Mix by gently flicking the tube (do not vortex) and centrifuge briefly.
14. Pipet DNAse I incubation mix (40 ul) directly onto PAXgene column and place upright at room temperature for 15 minutes.
15. Pipet 100 ul Buffer BR3 to the PAXgene column and centrifuge at 8000×g for 1 minute. Place the PAXgene column in a new 2 ml processing tube and discard the old processing tube containing flow through.
16. Apply 200 ul Buffer BR4 (Wash Buffer) to the PAXgene column and centrifuge for 1 minute at 8000×g. Place the PAXgene column in a new 2 ml processing tube and discard the old processing tube containing flow through. Note that the Buffer BR4 is supplied as a concentrate. Ensure that the ethanol is added to Buffer BR4 prior to use.
17. Add another 200 ul Buffer BR4 to the PAXgene column. Centrifuge for 3 minutes at 18000×g (max speed) to dry the PAXgene column membrane.
18. To eliminate residual Buffer BR4, discard the tube containing the flow through, place the PAXgene column in a 2 ml processing tube and centrifuge for 1 minute at full speed.
19. Discard the tube containing the flow through and transfer the PAXgene column to a 1.5 ml elution tube (from PAXgene kit). Pipet 30 ul Buffer BR5 directly on to the PAXgene column membrane (without touching the membrane with the pipet tip) and centrifuge for 2 minutes at 13000×g.
Data Analysis:
Differential Expression Analyses Across Patients
Samples were labeled “baseline” (stable RAPID3), “flare” (RAPID3 scores rose over two standard deviations above the baseline mean), or “steroid”. EdgeR (v3.24.3) (13) was used to analyze flare vs baseline differential gene expression. Permutation test (n=1×106) was used to test for the significance of overlap between genes decreased in flares in the index patient and patients 2, 3, and 4. GO enrichment (goana, from limma v3.38.3) (14) was used to identify enriched pathways in significantly differentially expressed genes in the index patient (FDR<0.1) and consistent in the direction of expression in both the index and replication patients (i.e., log fold change either both positive or both negative).
Time Series Analysis of Index Patient
We performed longitudinal data analysis on the index patient using ImpulseDE2 (v1.8.0) (15). Flare onset was defined clinically (as above) and samples from 8 weeks prior to flare up to 4 weeks after flare were analyzed (excluding any samples during which the patient was taking steroids, n=65 samples). The date of library preparation was included in the model for batch correction, and the genefilter (v1.64.0) package (16) was used to filter out lowly expressed genes. We hierarchically clustered mean expression of significantly differentially expressed genes by week to flare initiation (batch corrected logrpkm expression values were calculated using edgeR) and identified five coexpressed gene modules (Clusters 1-5). We analyzed these five modules for GO term enrichment (goana).
To compare differentially expressed gene modules over time, the mean expression level for each gene was calculated across flares per week, then normalized across weeks. ABIS (17) and CIBERSORTx (18) were used to deconvolute gene expression data. To aggregate a given cluster of genes or cell type with gene markers, the mean of standardized gene expression scores or deconvolved cell type scores, respectively, within each week were plotted. To identify synovial scRNAseq cluster specific marker gene signatures, we used a previously published dataset (18) to compare the cells from one scRNAseq cluster with cells from all the other scRNAseq clusters using the single-cell RNA-seq log2(CPM+1) matrix. We generated lists of the top 200 marker genes for each cluster using the criteria of 1) log2FC greater than 1, 2) auc greater than 0.6, and 3) percent of expressing cells greater than 0.4. We used Fisher's exact test to evaluate enrichment of synovial cell subtype marker genes in the 5 coexpressed gene modules.
Flow Cytometry and Sorting
Samples from PBMC were stained with antibodies to: CD31-APC, (WM59), Mouse IgG1-APC, (MOPC-21), PDPN-PerCP, (NZ1.3), Rat IgG2a, (eBR2a), CD45-PE, (HI30), Mouse IgG1-PE, (MOPC-21), TO-PRO®-3, and DAPI (4′,6-Diamidino-2-Phenylindole, Dihydrochloride). Cells were sorted on a BD FACSAria II for RNAseq. cDNA libraries were sequenced on MiSeq. DESeq2 (v1.24.0) (19) was used for differential expression analysis.
Statistics
R2 and Pearson correlation coefficients were calculated to assess the bivariate linear fit of disease activity measured by RAPID3 and DAS28 as well as CBC counts inferred from CIBERSORT cell counts and counts measured by clinical labs. Inferred CIBERSORTx lymphocyte counts were the sum of B cells naive+B cells memory+T cells CD8+T cells CD4 naive+T cells CD4 memory resting+T cells CD4 memory activated. One way ANOVA was used to test for significant differences among various clinical features according to disease activity state. Monocytes, Macrophages MO, Macrophages M1, and Macrophages M2 were summed to infer CIBERSORTx monocytes.
Results
Clinical Protocol Development
Four RA patients were followed for one to four years with weekly home collection of finger stick blood samples coupled with completion of RAPID3 and monthly clinic visits, where DAS28 were collected (
RNA was sequenced from a total of 189 finger stick blood samples from 4 patients, of which 162 (87%) passed quality control filtering.
We first assessed RNA quality and quantity by volume of fixative. 3 drops of blood were harvested with a 21 guage lancet and added to a microtainer tube prefilled with either 250 μl, 500 μl or 750 μl of PAX gene fixative. Samples were stored at room temperature for 3 days and then RNA was extracted using the PAX gene RNA kit and RIN scores and quantity of RNA was assessed using the Agilent 2100 Bioanalyzer picochip. RIN indicates the RNA integrity number which is an algorithm for assessing integrity values to RNA. The integrity of RNA is of significant importance for gene expression studies. RIN can and was traditionally evaluated using the 28S (˜5070 nucleotides) to 18S (˜1869 nucleotides) RNA ratio, which gives a ratio of about 2.7. A high 28S to 18S ratio is an indication that the purified RNA is intact and hasn't been degraded. RIN can easily be determined using Agilent 2100 Bioanalyzer measurements (Schroeder A et al (2006) BMC Mol Biol 7:3 (doi:10.1186/1471-2199-7-3). RNA samples should score RIN of >7 on a scale of 1 (highly degraded) to 10 (highest integrity). The results are depicted in
RNA integrity/quality and RNA quantity was evaluated from samples of 100 μl of blood in 250 μl PAX gene fixative with varying times of storage at room temperature (
RNA quality and quantity were evaluated in fresh and mailed samples (
RNA quality and quantity were evaluated by volume of extraction and washes (
RNA quality and quantity were evaluated from finger stick blood samples with RNA isolated using the PAXgeneRNA extraction versus a TriZol-based method. Mailed patient finger stick samples were stored in PAXgeneRNA buffer at −80° C. 142 samples had RNA extracted with PAXgeneRNA extraction with low volume washes, while 13 samples were thawed and mixed with 700 μl Trizol-LS and 250 μl chloroform. After centrifugation, the top layer was precipitated with isopropanol and glycogen and washed with 80% cold ethanol, centrifuged and the pellet was dried, resuspended in PBS and then purified using the Roche High Pure Isolation kit. RNA integrity and quality were both significantly reduced using Trizol and chloroform extraction versus the PAXgene RNA system. The Trizol reagent system utilizes guanidinium thiocyanate and phenol, and an organic extraction via phenol/chloroform.
Since ribosomal and hemoglobin RNA represent approximately 98% and 70% of the RNA in whole blood, respectively, we tested standard commercial kits for removing these RNAs prior to RNAseq. The PAXgene system does not remove globin mRNA, which can constitute up to 70% of the mRNA mass in whole blood total RNA. GlobinZero (Illumina) method and kit was utilized to remove globin mRNA from the samples. 4 ml heparinized blood was treated with 1 ug/ml LPS for one hour at 37° C. and 250 ul blood was placed into 250 μl PAXgene fixative in replicate microtainer tubes. After RNA extraction, samples were either treated with the globin zero depletion kit (globin and ribosomal depleted) or undepleted and then quantitative PCR was performed to test for hemoglobin A2, 18S RNA, or TNF alpha mRNA expression.
RNASeq QC metrics were assessed on RNA prepared with Illumina TruSeq or Kapa Hyper Prep Kits and having various RIN scores ranging from <5.7 to 8.1-10 (
To assess the validity of patient reported disease activity, we compared their RAPID3 scores with clinician collected DAS28. Significant correlations were evident between RAPID3 and DAS28 for each of the four patients (
Clinical and Molecular Features of RA Flare Compared to Baseline
Flares were associated with increases in objective clinical and laboratory measures of RA related disease activity in the index patient (
Time Series Analysis of Molecular Events Leading to RA Flares
To analyze the trajectories of gene expression over time and identify potential antecedents to flare, we performed time series analysis of the RNAseq data (
We further focused on two clusters that were differentially expressed antecedent to flare (
Antecedent cluster 3 (AC3) (Table 3) transcripts increased the week prior to flare and then decreased for the duration of flare (
Time Series Analysis of Synovial Cell Marker Genes in RA Flares
To better characterize the relevance of the clusters identified by the time series analysis to synovitis (
Overall, 622 of 625 AC3 genes decreased during flare in patient 1, and a subset (194 genes) also decreased in flares from at least 3 out of 4 RA patients (and 22 genes in 4 out of 4 patients;
We further tested whether cells that expressed surface markers of synovial fibroblasts were detectable in RA blood by flow cytometry. CD45−/CD31−/PDPN+ cells were increased in 19 additional RA patient blood relative to healthy controls (
The referenced TABLE 1 is provided below:
Discussion
We present longitudinal genomics as a strategy to study the antecedents to RA flare that may be generalizable to autoimmune diseases associated with waxing/waning clinical courses. We developed easy-to-use tools for patients to acquire both quantifiable clinical symptoms and molecular data at home over many years. This allowed us to capture data prior to the onset of clinical flares and retrospectively analyze it, identifying different RNA signatures (AC2 (Table 2) and AC3 (Table 3) evident in peripheral blood 1-2 weeks prior to flare.
The RNA signature of AC3 and sorted CD45−/CD31−/PDPN+ circulating cells revealed enrichment for pathways including cartilage morphogenesis, endochondral bone growth, and extracellular matrix organization (
Significantly, inflamed sublining fibroblasts are pathogenic in an animal model of arthritis (22). Our discovery that human AC3 genes share molecular characteristics of sublining fibroblasts, together with the observation that these cells spike prior to flare but are less detectable in blood during flare (
In addition, we observed a second RNA signature, AC2, activated in blood prior to the spike in AC3. AC2 bear RNA hallmarks of naive B cells. This finding is reminiscent of recent studies demonstrating autoreactive naive B cells are specifically activated in RA patients (24). While the triggers of these are unknown, infectious (for example bacterial or viral antigens), environmental or endogenous toxins (25-27) could provide a source of either specific antigens or activate pattern recognition receptors.
In conclusion, we demonstrate methods for densely collecting longitudinal clinical and gene expression data that can be used to discover changes in transcriptional profiles in the blood weeks prior to symptom onset. The methods include means and procedures for stabilizing, isolating and analyzing RNA from small volume samples which can be collected by a patient or individual themselves such as by finger stick collection, without the need for medical personnel, and which are applicable to home or field collection, to patients which are compromised or otherwise wherein collection of blood by venipuncture is not reasonable or available, and wherein there is a need for rapid sampling or for periodic sampling over time. This approach led to the identification and characterization of RNA markers and indicators of disease or pathological conditions and also the discovery of PRIME cells, bearing hallmarks of synovial fibroblasts, which are more common in RA patients and increase in blood just prior to flares. In modeling all our data, we suggest that prior to clinical flare, systemic B cell immune activation (detected as AC2) acts on PRIME cells, which traffic to the blood (detected as AC3) and subsequently to the synovial sublining during flares of disease activity.
More generally, application of an efficient self-collection protocol and approach combined with quantitative and qualitative RNA isolation to work in RA and RA patient sampling demonstrates the effectiveness and usefulness of our system. This initial study provides an exemplar of an approach to isolating, evaluating and assessing markers and RNA or protein expression changes and cellular changes which are applicable to disease assessment and evaluation, including in waxing/waning inflammatory disease, suggesting a general strategy relevant to numerous diseases and conditions, including additional disorders such as lupus, multiple sclerosis, and vasculitis.
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The system and methods detailed herein and in Example 1 provide a framework and approach to evaluating individuals at risk of disease, for assessing disease progression, or for identifying markers of more severe disease or metastasis. The availability of a system and method to isolate and analyze high quality RNA which validly identifies RNA, including RNA changes in vivo and new or altered RNAs such as those of different cellular components or of infectious agents, from patient self-collected samples, including small volume blood samples (finger stick), provides a means to monitor and assess various disease or infectious conditions or scenarios particularly where traditional blood sampling is not warranted, feasible or practical. Applications for evaluation and monitoring of cancer patients, including prior to and following treatment or remission, as well as patients with relapsing or remitting diseases such as multiple sclerosis, Crohn's disease, etc are contemplated. Further, the system and methods can be applied and implemented in infectious disease, including in viral diseases that affect large populations, either seasonally or in unanticipated circumstances. Those at risk of infection or who are presumed or determined to be infected can be evaluated to assess the RNA response and RNA indicators of disease, characterize predictors or markers of susceptibility or disease severity, and identify targets for treatment or modulation. For example, more precise and marker-based knowledge and understanding of influenza virus infection and susceptibility could reduce the effects of seasonal influenza on individuals and the health care system. Further, the recent outbreak of new coronavirus SARS-COV2 and the COVID-19 pandemic underscores an imminent need for a system, method and approach as provided herein.
Coronaviruses are a family of viruses that can cause illnesses such as the common cold, severe acute respiratory syndrome (SARS) and Middle East respiratory syndrome (MERS). In late 2019, a new coronavirus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), was identified as the cause of a COVID-19 disease outbreak that originated in China. In March 2020, the World Health Organization (WHO) declared the COVID-19 outbreak a pandemic. By early April, the worldwide number of confirmed COVID-19 cases were nearly 1 5 million, with over 400,000 in the U.S., over 135,000 cases in each of Italy and Spain, over 100,000 cases in Germany and over 80,000 reported in China. Deaths worldwide were over 80,000. No accepted or approved treatments or vaccines are available.
Signs and symptoms of COVID-19 appear 2 to 14 days after exposure and can include fever, cough, shortness of breath or difficulty breathing, as well as tiredness, aches, runny nose and sore throat. Some people experience the loss of smell or taste. People who are older or who have existing chronic medical conditions, such as heart disease, lung disease or diabetes, or who have compromised immune systems may be at higher risk of serious illness, similar to what is seen with other respiratory illnesses, such as influenza.
The severity of COVID-19 symptoms can range from very mild to severe and some people may have no symptoms at all. In fact, studies have shown that a significant portion of individuals with coronavirus lack symptoms (“asymptomatic”) and that even those who eventually develop symptoms (“pre-symptomatic”) can transmit the virus to others before showing symptoms (Li R et al Science 10.1126/science.abb3221(2020); Rothe C et al (2020) New Engl J Med 382(10):970-971; Zou L et al (2010) New Engl J Med 382(12)1177-1179). Therefore, the virus can spread between people interacting in close proximity—for example, speaking, coughing, or sneezing—even if those people are not exhibiting symptoms.
In the United States, nearly one-third of COVID-19 disease cases are 6 or older and patients over 65 account for nearly half of hospitalizations and a significant majority of deaths, according to CDC reports. Nonetheless, about 20% of infected people ages 20-44 are hospitalized, demonstrating that this is not just a disease of older adults. Important outstanding questions exist as to the underlying biological vulnerability of older individuals and how do preexisting conditions or illnesses exacerbate COVID-19. Also, it would be helpful to have indicators for those patients who will develop more significant or severe disease, so they can be managed or triaged differently or more aggressively.
RNA monitoring and longitudinal genomics in accordance with the system and methods provided herein, including as set out in Example 1, provides an approach to isolate, identify and evaluate RNAs in individuals exposed to or at risk of virus infection, such as coronavirus infection, such as with SARS-COV2, or patients infected with the virus and diagnosed for COVID-19. The systems and methods could be implemented in individuals post-vaccine also to evaluate RNA, protein and cellular response(s). Finger stick collection of small blood samples as described herein may be implemented by regular collection at home, at or in hospital, by medical care workers or personnel, or in isolation or quarantine. This permits monitoring of the infection, including viral RNA, disease, RNA response, RNA alterations, including as an indicator of cellular response as described above and in Example 1. The availability of high quality RNA from prospective and retrospective sampling will facilitate an understanding of infection and disease, including in influenza, coronavirus, or instances of other known or unknown infectious agents, including new variants, as well as the body's response to disease and susceptibility to disease aspects. Collection of standard venipuncture samples puts health care workers at risk and is unduly invasive and difficult for patients and individuals already suffering or in stressful and demanding situations and conditions.
This invention may be embodied in other forms or carried out in other ways without departing from the spirit or essential characteristics thereof. The present disclosure is therefore to be considered as in all aspects illustrated and not restrictive, the scope of the invention being indicated by the appended Claims, and all changes which come within the meaning and range of equivalency are intended to be embraced therein.
Various references are cited throughout this Specification, each of which is incorporated herein by reference in its entirety.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US2021/034785 | 5/28/2021 | WO |
| Number | Date | Country | |
|---|---|---|---|
| 63031861 | May 2020 | US | |
| 63050155 | Jul 2020 | US | |
| 63135159 | Jan 2021 | US | |
| 63135224 | Jan 2021 | US | |
| 63171749 | Apr 2021 | US |
