Methods for localized in situ detection of mRNA

• US 8,551,708 B2
• Filed: 02/15/2012
• Issued: 10/08/2013
• Est. Priority Date: 02/15/2011
• Status: Active Grant

• Current Assignee: Leica Biosystems Newcastle Ltd. (Danaher Corporation)
• Original Assignee: Leica Biosystems Newcastle Ltd. (Danaher Corporation)
• Inventors: Bernitz, Mats Nilsson, Larsson, Chatarina, Grundberg, Ida
• Primary Examiner(s): WOOLWINE, SAMUEL C
• Application Number:
  US13/397,503
• Publication Number:
  US 20120270214A1
• Time in Patent Office: 601 Days
• Field of Search: None
• US Class Current: 435/6.12
• CPC Class Codes: C12Q 1/6809 Methods for determination or identification of nucleic acids involving differential detection
• CPC Class Codes: C12Q 1/6827 for detection of mutation or polymorphism
• CPC Class Codes: C12Q 1/6841 In situ hybridisation
• CPC Class Codes: C12Q 1/6886 for cancer immunoassay for cancer G01N33/574
• CPC Class Codes: C12Q 2521/107 RNA dependent DNA polymerase,(i.e. reverse transcriptase)
• CPC Class Codes: C12Q 2521/327 RNAse, e.g. RNAseH
• CPC Class Codes: C12Q 2525/107 incorporating a peptide nucleic acid
• CPC Class Codes: C12Q 2525/125 incorporating agents resulting in resistance to degradation
• CPC Class Codes: C12Q 2525/161 incorporating target specific and non-target specific sites
• CPC Class Codes: C12Q 2525/307 Circular oligonucleotides
• CPC Class Codes: C12Q 2531/125 Rolling circle
• CPC Class Codes: C12Q 2537/143 Multiplexing, i.e. use of multiple primers or probes in a single reaction, usually for simultaneously analyse of multiple analysis
• CPC Class Codes: C12Q 2543/10 the purpose being "in situ" analysis
• CPC Class Codes: C12Q 2600/112 Disease subtyping, staging or classification
• CPC Class Codes: C12Q 2600/156 Polymorphic or mutational markers
• CPC Class Codes: C12Q 2600/16 Primer sets for multiplex assays

Abstract

The present invention relates to the detection of RNA in a sample of cells. More particularly, the present invention relates to the localized detection of RNA in situ. The method relies on the conversion of RNA to complementary DNA prior to the targeting of the cDNA with a padlock probe(s). The hybridization of the padlock probe(s) relies on the nucleotide sequence of the cDNA which is derived from the corresponding nucleotide sequence of the target RNA. Rolling circle amplification of the subsequently circularized padlock probe produces a rolling circle product which may be detected. Advantageously, this allows the RNA to be detected in situ.

Claims

1. A method for localized in situ detection of mRNA which codes for one or more mutations of the KRAS gene in a sample of cells that were formalin-fixed paraffin-embedded (FFPE) on a slide surface, comprising:

(a) generating cDNA from mRNA in the sample using a primer;

(b) digesting the mRNA hybridized to said cDNA;

(c) contacting said sample with one or more padlock probes specific for mutations to the KRAS gene;

(d) joining, directly or indirectly, the ends of said padlock probe(s) to form circularized padlock probe(s);

(e) subjecting said circularized padlock probe(s) to rolling circle amplification (RCA) to generate rolling circle amplification products; and

,

(f) detecting the rolling circle amplification product(s), wherein the padlock probe comprises;

(i) Y1-X1-Z1-A

(ii) Y1-X1-Z1-T

(iii) Y1-X1-Z1-C

(iv) Y2-X1-Z2-A

(v) Y2-X1-Z2-T

(vi) Y2-X1-Z2-C

(vii) Y3-X1-Z3-A;

(viii) Y1-X2-Z1-G

(ix) Y2-X2-Z2-G, or

(x) Y3-X2-Z3-G

where;

X1 or X2 is from 16-50 nucleotides;

Y1+Z1=20 to 29 nucleotides;

Y2+Z2=20 to 29 nucleotides;

Y3+Z3=20 to 29 nucleotides;

Y1 is GTGGCGTAGGCAAGA (SEQ ID NO;

1), GTGGCGTAGGCAAG (SEQ ID NO;

2), GTGGCGTAGGCAA (SEQ ID NO;

3), GTGGCGTAGGCA (SEQ ID NO;

4), GTGGCGTAGGC (SEQ ID NO;

5), GTGGCGTAGG (SEQ ID NO;

6), GTGGCGTAG, GTGGCGTA, GTGGCGT, or GTGGCG;

Y2 is TGGCGTAGGCAAGAG (SEQ ID NO;

7), TGGCGTAGGCAAGA (SEQ ID NO;

8), TGGCGTAGGCAAG (SEQ ID NO;

9), TGGCGTAGGCAA (SEQ ID NO;

10), TGGCGTAGGCA (SEQ ID NO;

11), TGGCGTAGGC (SEQ ID NO;

12), TGGCGTAGG, TGGCGTAG, TGGCGTA, or TGGCGT;

Y3 is CGTAGGCAAGAGTGC (SEQ ID NO;

13), CGTAGGCAAGAGTG (SEQ ID NO;

14), CGTAGGCAAGAGT (SEQ ID NO;

15), CGTAGGCAAGAG (SEQ ID NO;

113), CGTAGGCAAGA (SEQ ID NO;

114), CGTAGGCAAG (SEQ ID NO;

115), CGTAGGCAA, CGTAGGCA, CGTAGGC, or CGTAGG;

Z1 is TGGTAGTTGGAGCT (SEQ ID NO;

27), GGTAGTTGGAGCT (SEQ ID NO;

28), GTAGTTGGAGCT (SEQ ID NO;

29), TAGTTGGAGCT (SEQ ID NO;

30), AGTTGGAGCT (SEQ ID NO;

31), GTTGGAGCT, TTGGAGCT, TGGAGCT, GGAGCT, or GAGCT;

Z2 is GGTAGTTGGAGCTG (SEQ ID NO;

16), GTAGTTGGAGCTG (SEQ ID NO;

17), TAGTTGGAGCTG (SEQ ID NO;

18), AGTTGGAGCTG (SEQ ID NO;

19), GTTGGAGCTG (SEQ ID NO;

20), TTGGAGCTG, TGGAGCTG, GGAGCTG, GAGCTG, or AGCTG; and

Z3 is AGTTGGAGCTGGTG (SEQ ID NO;

21), GTTGGAGCTGGTG(SEQ ID NO;

22), TTGGAGCTGGTG(SEQ ID NO;

23), TGGAGCTGGTG(SEQ ID NO;

24), GGAGCTGGTG(SEQ ID NO;

25), GAGCTGGTG(SEQ ID NO;

26), AGCTGGTG, GCTGGTG, CTGGTG, or TGGTG.

2. The method of claim 1, wherein generating the cDNA complementary to the RNA in the sample comprises contacting said sample with a reverse transcriptase and a reverse transcription primer.

3. The method of claim 2, wherein the reverse transcription primer is ribonuclease resistant.

4. The method of claim 2, wherein the reverse transcription primer is modified so as to be capable of immobilization in said cells.

5. The method of claim 4, wherein the reverse transcription primer has a functional moiety capable of binding to or reacting with a cell or cellular component or an affinity binding group capable of binding to a cell or cellular component.

6. The method of claim 1, comprising adding a ribonuclease to the sample.

7. The method of claim 1, wherein the collection of padlock probes comprise different detection probe binding regions.

8. The method of claim 7, where the different detection probe binding regions are recognized by differently labeled detection probes, wherein the detection probes comprise different fluorescent labels, chromogenic labels, radioactive labels, luminescent labels, magnetic labels, or electron-density labels.

9. The method of claim 1, wherein X2 differs from X1.

10. The method of claim 1, wherein the primer comprises 2′

O-Me RNA, methylphosphonates or 2′

Fluor RNA bases, peptidyl nucleic acid residues, or locked nucleic acid residues.

11. The method of claim 10, wherein the primer comprises one or more locked nucleic acid residues.

12. The method of claim 11, wherein the primer comprises 2 or more locked nucleic acids separated by 1 or more natural or synthetic nucleotides in the primer sequence.

13. The method of claim 1, further comprising adding a ribonuclease to digest mRNA hybridized to said cDNA.

14. The method of claim 13, wherein the ribonuclease is RNase H.

15. The method of claim 1, wherein X1 and X2 each comprise at least one labeled nucleotide.

16. The method of claim 15, wherein the labeled nucleotide comprises a fluorophore or a chromophore.

17. The method of claim 1, wherein each probe selected from (i)-(vii) has the same X1.

18. The method of claim 1, wherein each probe selected from (viii)-(x) has the same X2.

19. The method of claim 1, wherein the rolling circle amplification uses a DNA polymerase having 3′

-5′

exonuclease activity capable of digesting the cDNA to generate a free 3′

end which acts as a primer for said RCA.

20. The method of claim 19, wherein the DNA polymerase is a Φ

29 polymerase.

21. The method of claim 1, further comprising contacting the sample with an exonuclease to digest the cDNA to generate a free 3′

end that acts as a primer for RCA.

22. The method of claim 1, wherein in the contacting step (c), the sample is contacted with at least a first and a second padlock probe, wherein the first padlock probe comprises terminal regions complementary to immediately adjacent regions on the cDNA, and wherein the second padlock probe comprises terminal regions that differ from the terminal regions of the first padlock probe only by a single nucleotide at the 5′

or 3′

terminus of the second padlock probe.

23. The method of claim 1, wherein multiple different mRNAs are detected using multiple different padlock probes.

24. The method of claim 1, wherein mRNA is detected in a single cell.

25. The method of claim 1, wherein the sample is derived from a tissue or organ of the body, or from a bodily fluid.

26. The method of claim 1, wherein the sample is a colon, lung, pancreas, prostate, skin, thyroid, liver, ovary, endometrium, kidney, brain, testis, lymphatic fluid, blood, plasma, urinary bladder, or breast sample.

27. The method of claim 1, wherein the sample is suspected to comprise an mRNA found in a cancer cell.

28. The method of claim 1, wherein the one or more padlock probes has a GC content of at least 40%.