Methods for localized in situ detection of mRNA
First Claim
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).
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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.
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Citations
25 Claims
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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:
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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). - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13)
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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:
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(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. - View Dependent Claims (15, 16, 17)
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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:
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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. - View Dependent Claims (19, 20, 21, 22, 23)
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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:
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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. - View Dependent Claims (25)
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Specification