Methods for localized in situ detection of mRNA

• US 8,551,710 B2
• Filed: 07/05/2012
• 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:
  US13/542,378
• Publication Number:
  US 20130004953A1
• Time in Patent Office: 460 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 at least one target RNA in a sample of one or more cells that was formalin-fixed paraffin-embedded (FFPE), comprising:

generating a cDNA complementary to an RNA in the sample;

adding a ribonuclease to said sample to digest the RNA hybridized to said cDNA;

contacting said sample with one or more padlock probes wherein said padlock probe(s) comprise terminal regions complementary to said cDNA and hybridizing said padlock probe to the cDNA at said complementary terminal regions;

circularising said padlock probe(s) by ligating, directly or indirectly, the ends of said padlock probe(s);

subjecting said circularized padlock probe(s) to rolling circle amplification (RCA);

detecting the rolling circle amplification product(s).

2. The method of claim 1 wherein the RNA is mRNA.

3. 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.

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

5. The method of claim 4, wherein the reverse transcription primer comprises 2′

O-Me RNA, methylphosphonates or 2′

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

6. The method of claim 5, wherein the reverse transcription primer comprises one or more locked nucleic acid residues.

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

end which acts as a primer for said RCA.

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

or 3′

terminus of said second padlock probe.

9. The method of claim 8, wherein said first padlock probe comprises a first detection probe binding region, and said second padlock probe comprises a second detection probe binding region.

10. The method of claim 9, further comprising contacting said sample with at least a first labeled detection probe comprising a sequence identical to the first detection probe binding region of the first padlock probe, and a second labeled detection probe comprising a sequence identical to the second detection probe binding region of the second padlock probe, and hybridizing said first and second labeled detection probes to the rolling circle amplification products.

11. The method of claim 10, further comprising detecting a signal from said first labeled detection probe hybridized to the rolling circle amplification product, or detecting a signal from said second labeled detection probe hybridized to the rolling circle amplification product, or detecting a signal from both said first and second labeled detection probes hybridized to the rolling circle amplification products, wherein the detection of said first labeled detection probe indicates the presence of wild-type mRNA in the cell and the detection of said second labeled detection probe indicates the presence of variant mRNA in the cell.

12. The method of claim 1, wherein multiple different RNAs are detected using multiple different padlock probes.

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

14. 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 was 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.

15. The method of claim 14 further comprising:

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

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

(f) detecting the rolling circle amplification product(s).

16. The method of claim 14, wherein the primer comprises 2′

O-Me RNA, methylphosphonates or 2′

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

17. The method of claim 14, wherein multiple different KRAS mutations are detected using multiple different padlock probes.

18. A method for determining the presence and/or location of a genetic sequence in a cell in a biological sample that was formalin-fixed paraffin-embedded (FFPE) comprising:

a) hybridizing a DNA complement having the genetic sequence to RNA in the biological sample;

b) digesting RNA hybridized to the DNA complement;

c) hybridizing a first padlock probe to at least a portion of the DNA complement, wherein the padlock probe comprises the genetic sequence on one of two terminal ends that are complementary to different but immediately adjacent regions of the DNA complement;

d) joining the two terminal ends of the padlock probe to form a circularized probe;

e) replicating the circularized probe to yield a nucleic acid molecule comprising multiple copies of the replicated probe; and

f) detecting presence, absence, and/or location of the genetic sequence in the cell using a probe that hybridizes to the nucleic acid molecule.

19. The method of claim 18, further comprising generating the DNA complement.

20. The method of claim 19, wherein the DNA complement is generated using a primer that is ribonuclease resistant.

21. The method of claim 18, wherein the circularized probe is replicated using a polymerase having 3′

-5′

exonuclease activity.

22. The method of claim 21, wherein the circularized probe is incubated under conditions to generate a free 3′

end on the DNA complement that acts as a primer for replication of the circularized probe.

23. The method of claim 18, further comprising adding a second padlock probe that is complementary to a second genetic sequence.

24. A method for in situ detection and/or localization of a specific RNA in a cell from a formalin-fixed paraffin-embedded (FFPE) biological sample on a solid support comprising:

a) incubating an immobilized FFPE biological sample on solid support with reverse trancriptase and a ribonuclease-resistant primer under conditions to generate a DNA molecule complementary to the specific RNA to form an RNA-DNA hybrid;

b) adding a ribonuclease and incubating the ribonuclease under conditions to digest RNA in the RNA-DNA hybrid;

c) incubating the digested RNA-DNA hybrid under conditions to hybridize a complementary padlock probe to the DNA portion of the digested RNA-DNA hybrid, wherein the padlock probe comprises two terminal ends that are complementary to different but immediately adjacent regions of the DNA molecule;

d) incubating the padlock probe hybridized to the DNA portion of the RNA-DNA hybrid with a ligase under conditions to ligate the terminal ends of the padlock probe;

e) incubating the ligated padlock probe with a polymerase and nucleotides under conditions to create a primer from the DNA that is used to replicate the padlock probe and generate a nucleic acid comprising multiple copies of the replicated padlock probe; and

,

f) incubating the nucleic acid with one or more complementary nucleic acid probes to detect the presence, absence, or location of the specific RNA in the cell.

25. The method of claim 24, further comprising adding a second padlock probe that is complementary to a second specific RNA.