Patent Application
20200141928
The present invention relates to a method for RNA tagging and analysis on single cell, suitable for platelets and cells with a short half-life, comprising the following steps:
Platelets are corpuscolar blood elements playing a crucial role in coagulation. The crucial role played by platelets in coagulation makes them key players in thrombotic phenomena. Recently, other functions have been attributed to platelets, such as, vascular integrity control, involvement in inflammatory and immune processes, tumour metastasis, angiogenesis and, last but not least, the onset and progression of atherothrombotic disease. The characteristic which distinguishes platelets is that they are core-free. Conversely, platelets possess granules, cytoplasmic organelles, and RNA.
Given the multifaceted role played by platelets and the presence of RNA therein, the need for tagging and analyzing the RNA contained therein is strongly felt.
The SmartFlare™ RNA detection probe technology (Merck Millipore, Germany) allows for the analysis of intracellular RNA expression on single living cell. The SmartFlare™ method includes plating the cells, preferably in 96-well plates, at a 60-80% confluence. The SmartFlare™ probe is added to the culture and allowed to incubate for a period of about 16 hours, so as to allow it to enter the cells via endocytosis. Once inside the cell, the probe, recognizing a specific target, binds thereto and a subsequent fluorescence analysis reveals the presence thereof. Since platelets, as well as other cells with short half-life, such as monocytes, lymphocytes, granulocytes, may not be maintained in culture for a time as long as that required by the method, they are not suitable for the analysis by means of the above method.
The first attempts to introduce nucleic acids into platelets have been described by Hong W. et al. Transfection of Human Platelets with Short Interfering RNA, Clin Transl Sci. 2011; 4 (3): 180-182, though achieving a very low efficiency, equal to about 9%. Method improvements are described in WO 2014118817, with the purpose of using siRNA in platelets. However, no attempt is described about platelet RNA tagging, in particular, mRNA and miRNA, which tagging is thus not readily obtainable with the technologies available to date. In particular, a strong need for tagging RNA species modulated in acute pathology conditions is felt. Therefore, a method which allows to tag platelet RNA and cells with short half-life in vitro is needed, in order to overcome the limits imposed by the characteristics of the platelets themselves.
The present invention describes a method which surprisingly allows to overcome the limits imposed by the particular nature of platelets and cells with short half-life, thus leading to an efficient tagging of platelet RNA and cells with short half-life in vitro.
It is herein described an innovative method for RNA tagging and analysis on single cell, suitable for platelets and cells with a short half-life, comprising the following steps:
In a preferred embodiment, said RNA is mRNA or miRNA.
For the aim of the present invention, SmartFlare™ probes mean probes provided by Merck Millipore, which recognize mRNA or miRNA key target. Said probes are Cy5 or Cy3 tagged. However, by means of the methods known to those skilled in the art, further probes may be synthesised for specific purposes, i.e. probes recognizing targets of particular interest. For the aim of the present invention, SmartFlare™ probes are probes capable of entering a cell, recognizing a target RNA of interest, and making it detectable by the SmartFlare™ method; by way of not limiting example, the SmartFlare™ probes found in the Merck Millipore catalogue are included. Also included are those additional probes which may be synthesised by those skilled in the art for further targets and purposes of interest, in particular oligonucleotide sequences capable of pairing with target RNAs, such as by way of example DNA sequences, tagged with a fluorophore detectable by cytofluorimetry and microscopy.
In a preferred embodiment, said method is operated on platelets. Preferably, said incubation takes place for about 1 hour at room temperature, or at about 37° C. Preferably, said culture medium is RPMI1640 supplemented with glutamine or GlutaMAX™.
In a still more preferred embodiment, about 500,000 platelets resuspended in about 100 μl of culture medium are provided. Preferably, said lipofection reagent is selected from: TransIT-LT1 (Mirus), Turbofect transfection reagent (Invitrogen), RiboJuice™ siRNA Transfection Reagent (Merck Millipore), NanoJuice® Transfection Kit (Merck Millipore), GeneJuice® Transfection Reagent (Merck Millipore) and other transfecting reagents known to those skilled in the art. The experimental data obtained and reported below showed that, despite the great issues, well known in the prior art, relating to the introduction of nucleic acids in platelets, it was surprisingly possible to achieve more than satisfactory transfection levels using TransIT-LT1 (Mirus), Gene Juice, Nano Juice, Ribo Juice (Merck Millipore) and Turbofect transfection reagent (Invitrogen). From a morphological analysis, it has been observed that, upon transfection, the platelets do not exhibit major alterations.
1.5 μl of said lipofection reagents, 1:10 diluted, are added to 100 μl of the culture medium. Preferably, said culture medium contains 300 pM of said SmartFlare™ probe.
Preferably, said fixative used in said step c) is 1% paraformaldehyde (PFA).
Optionally, at the end of the incubation as described in step b) and before said fixing step c), said platelets are tagged with a specific antibody, for example FITC-tagged antiCD41. Thereby, platelets containing the RNA of interest may be detected.
It is particularly efficient, for the purposes of the present invention, isolating platelets from whole blood by the method described below. The blood is collected by venous withdrawal from the cephalic vein of donors who have signed their informed consent to participate in the study. Whole blood (WB) was taken by means of a 19-gauge needle, without venous stasis, and placed in a tube containing citrate ( 1/10 of 0.129 M sodium citrate volume) and corn trypsin inhibitor (50 μg/ml) (Vacutainer, Becton Dickinson) discarding the first 4 ml. For platelet preparation, WB was centrifuged at room temperature, in the absence of a brake, preferably at 100 g for 15′. For the aim of the present invention, the total platelet preparation analysed by the Sysmex XE-2100 Automated Hematology Analyzer is used to determine the platelet recovery, the platelet average volume (MPV), the platelet immature fraction (IPF), and the platelet distribution width (PDW).
Some examples of results obtained by the method according to the present invention are shown hereinbelow. Said examples are not intended to limit in any manner the scope of the present invention to that specifically exemplified herein. In particular, the results obtained using probes for the Tissue Factor (TF mRNAs) and 18S in platelets are herein described. However, probes for further RNAs of specific interest may be synthesised and used according to methods known to those skilled in the art.
A platelet preparation was incubated with a SmartFlare™ probe for the uptake control. Each sample was incubated in the presence of one of the lipofection reagents shown in
The results are shown in
It is known that a subpopulation of human platelets express TF mRNA. The used probes include:
The platelets were isolated from whole blood of healthy volunteers after signature of the informed consent, according to methods known to those skilled in the art.
For each of the above probes, 500,000 platelets were incubated for 1 hour at room temperature in RPMI1640 culture medium supplemented with glutamine with 1.5 μl of transfection reagent Transit-LT1 1:10 diluted and 300 pM of one of the above SmartFlare™ probes.
After said incubation, the platelets were tagged with a FITC-tagged anti-CD41 antibody and fixed with 1% PFA. By cytofluorimetry, the Cy5 fluorescence of the probes was analysed in the CD41 positive platelets.
Once the platelet aggregates have been excluded, the signal from the positive control (Uptake Cy5) and the negative control (Scramble Cy5) was assessed. By means of the Kaluza analysis software, the median fluorescence intensity (MFI) of the probes which respectively detect 18S and TF mRNAs between the CD41 and Cy5 positive events was quantified, using Scramble Cy5 as the negative control to subtract the unspecified signal. The data are expressed as MFI and the results are shown in
| Number | Date | Country | Kind |
|---|---|---|---|
| 102016000064945 | Jun 2016 | IT | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/IB2017/053733 | 6/22/2017 | WO | 00 |
