Parallel microarray hybridization

Abstract

A glass substrate with multiple identical microarrays is provided, for example, for the identification of genes via nucleic acid hybridization. The multiarray substrate permits the analysis, in parallel, of several different samples on the same substrate and thus under the same conditions. Results obtained using the multiarray substrate are therefore less variable than those obtained with conventional techniques.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Schematic representation of a glass slide with multiple parallel microarrays.

FIG. 2. Flow diagram of the steps of the method of the invention.

FIGS. 3A-E. Reproducibility of hybridizations. (A-C): typical scatter plots of self-self hybridization of lung cDNAs between two channels within a block (within-block, panel A), two different blocks in one slide (within-slide, panel B), and among slides (among-slide, panel C), respectively. The cDNAs from an identical lung tissue were labeled with Cy3 or Alexa 647, and hybridized to each block of the slides. The numbers on x- and y-axis were background-subtracted fluorescence intensities of each spot with log 2 transformation. (D) A comparison of correlation coefficients from replicated hybridizations. The results were expressed as means±SE. *P<0.01 v.s. among-slide; #P<0.01 v.s. within-slide. (E) Comparison of accumulated errors between within-slide and among-slide groups. For the within-slide group, the log ratios were from parallel hybridization on a single slide. For the among-slides, the log ratios were from different slides. The accumulated errors were calculated as described in Materials and Methods.

FIG. 4. Summary of differentially expressed genes among 6 organs. The number under an organ represents the genes that are expressed significantly higher in the respective organ compared to other organs (p<0.05). Similarly, the number between any two organs represents the genes that are expressed significantly higher in the two organs compared to other organs (p<0.05). Thicker lines highlight a larger number of the genes co-expressed in the respective two organs.

FIGS. 5A and B. Hot maps of Organ-prominent genes. Left (A) and right (B) panels are the relative expression levels of genes differentially expressed in one and two organs, respectively. Each column represents 19 replicated hybridizations of each organ and each row shows the spot signals of the organ-prominent genes. The scale of normalized spot signals was indicated on the top of the graph. (A): lung: 166 genes; (B) heart: 100 genes; (C) kidney: 186 genes; (D) liver: 324 genes; (E) spleen: 88 genes; (F) brain: 225 genes; (G) lung-heart: 47 genes; (H) lung-liver: 33 genes; (I) lung-spleen: 95 genes; (J) kidney-liver: 174 genes; (K) lung-kidney: 21 genes; (E) kidney-brain: 21 genes.

FIG. 6. Relative mRNA abundance of lung-prominent genes determined by relative real-time PCR The mRNAs from six organs were reverse-transcribed to cDNA and quantified by relative real-time PCR. All of the genes were run on the same plate with 18S rRNA as an endogenous reference. The results were expressed as % of lung. Data shown are means±S.E. (n=3 biological replications).The mRNA expression level of all the genes in the lung was significantly higher in other organs (P<0.05).

FIG. 7 depicts DNA microarray signal intensities and spot images for 13 verified genes in tabular form.

FIG. 8 is a schematic representation of an alternative embodiment of the invention where identical sets of different microarrays are present on the single glass substrate.

Claims

1. A hybridization system, comprising a single glass substrate;a plurality of microarrays, wherein said microarrays are separated from one another; andbinding entities for binding one or more substances in one or more samples, said binding entities forming at least a part of each of said plurality of microarrays.

2. The hybridization system of claim 1, wherein said glass substrate is a glass microscope slide.

3. The hybridization system of claim 1, wherein each of said microarrays in said plurality of microarrays includes identical binding entities.

4. The hybridization system of claim 1, wherein said binding entities include nucleic acid.

5. The hybridization system of claim 4, wherein said nucleic acid is DNA.

6. The hybridization system of claim 1, wherein said plurality of microarrays is attached to said single glass substrate by printing.

7. The hybridization system of claim 1, further comprising a barrier between each microarray in said plurality of microarrays.

8. A method of producing a hybridization system, comprising the step of printing a plurality of microarrays on a single glass substrate, wherein said microarrays are separated from one another, and wherein at least a part of each of said microarrays includes one or more binding entities.

9. The method of claim 8, wherein said single glass substrate is a glass microscope slide.

10. The method of claim 8, wherein each of said plurality of microarrays includes identical binding entities.

11. The method of claim 8, wherein each of said binding entities include nucleic acid.

12. The method of claim 11, wherein said nucleic acid is DNA.

13. The method of claim 8, further comprising the step of forming a barrier between each microarray of said plurality of microarrays.

14. A method of comparing, on a single substrate, hybridization patterns of molecules in a plurality of samples, comprising the steps of exposing each microarray of a plurality of microarrays formed on a single glass substrate and separated from one another to a) one sample of said plurality of samples; orb) two or more samples of said plurality of samples, wherein said two or more samples are differentially labeled; anddetecting hybridization patterns of molecules in said plurality of samples.

15. The method of claim 14, wherein said single glass substrate is a glass microscope slide.

16. The method of claim 14, wherein said plurality of microarrays are identical.

17. The method of claim 14, wherein said plurality of microarrays comprise nucleic acid.

18. The method of claim 17, wherein said nucleic acid is DNA.

19. The method of claim 14, wherein said plurality of microarrays is attached to said single glass substrate by printing.

20. The method of claim 14, wherein a barrier if formed between each microarray in said plurality of microarrays.