Universal amplification of fragmented RNA

Abstract

The invention relates to methods of using fragmented RNA, such as RNA obtained from archived fixed paraffin-embedded tissue material (FPET RNA) or other clinically biopsied tissue specimens for universal gene expression profiling.

1 . A method for preparing fragmented RNA comprising a multiplicity of RNA species for gene expression analysis comprising the steps of: 
 (a) polyadenylating the fragmented RNA, and    (b) converting the polyadenylated fragmented RNA obtained in step (a) to cDNA.    
     
     
         2 . The method of  claim 1  wherein the size of the RNA species within the fragmented RNA is between about 20 bases and about 2000 bases.  
     
     
         3 . The method of  claim 1  wherein the average size of the RNA species within the fragmented RNA is about 50 and about 300 bases.  
     
     
         4 . The method of  claim 1  wherein polyadenylation is performed with  E. coli  polyA polymerase.  
     
     
         5 . The method of  claim 1  additionally comprising the step of deblocking the 3′ termini of the fragmented RNA species prior to step (a) with a deblocking agent.  
     
     
         6 . The method of  claim 5  wherein said deblocking agent is a phosphatase enzyme.  
     
     
         7 . The method of  claim 6  wherein said phosphatase is selected from the group consisting of calf alkaline phosphatase (CIP), bacterial alkaline phosphatase, shrimp alkaline phosphatase, and variants thereof.  
     
     
         8 . The method of  claim 5  wherein said deblocking agent is a polynucleotide kinase (PNK), or a variant thereof.  
     
     
         9 . The method of  claim 8  wherein said polynucleotide kinase is T4 polynucleotide kinase (T4 PNK), or a variant thereof.  
     
     
         10 . The method of  claim 5  wherein said deblocking agent is removed prior to performing the polyadenylation step (a).  
     
     
         11 . The method of  claim 5  wherein polyadenylation step (a) is performed without prior removal of the deblocking agent.  
     
     
         12 . The method of  claim 11  wherein said deblocking agent is a polynucleotide kinase (PNK).  
     
     
         13 . The method of  claim 1  wherein the polyadenylated fragmented RNA obtained in step (a) is converted to cDNA by treatment with a reverse transcriptase and oligo-dT primers.  
     
     
         14 . The method of  claim 13  wherein said reverse transcriptase is selected from the group consisting of avian myeloblastosis virus reverse transcriptase (AMV-RT), Moloney murine leukemia virus reverse transcriptase (MMLV-RT), and recombinant heterodimeric reverse transcriptases expressed in  E. coli.    
     
     
         15 . The method of  claim 14  wherein said oligo-dT primers contain an RNA polymerase promoter sequence.  
     
     
         16 . The method of  claim 15  wherein said promoter is a T7 RNA polymerase promoter.  
     
     
         17 . The method of  claim 13  wherein said polyadenylated fragmented RNA obtained in step (a) is immobilized prior to conversion to cDNA.  
     
     
         18 . The method of  claim 17  wherein said immobilization is performed on beads.  
     
     
         19 . The method of  claim 18  wherein the immobilized polyadenylated fragmented RNA is enriched prior to conversion to cDNA.  
     
     
         20 . The method of  claim 19  wherein the enrichment comprises removal of rRNA sequences by hybridization to bead immobilized complementary rRNA oligonucleotides.  
     
     
         21 . The method of  claim 13  wherein the RNA is mRNA obtained from a fixed, paraffin-embedded tissue sample.  
     
     
         22 . The method of  claim 21  wherein said tissue sample is from a tumor.  
     
     
         23 . The method of  claim 22  wherein said tumor is cancer.  
     
     
         24 . The method of  claim 23  wherein said cancer is selected from the group consisting of breast cancer, colon cancer, lung cancer, prostate cancer, hepatocellular cancer, gastric cancer, pancreatic cancer, cervical cancer, ovarian cancer, liver cancer, bladder cancer, cancer of the urinary tract, thyroid cancer, renal cancer, carcinoma, melanoma, and brain cancer.  
     
     
         25 . The method of  claim 15  further comprising the step of (c) PCR amplification using one or more cDNA species present in the cDNA obtained in step (b) as a template.  
     
     
         26 . The method of  claim 25  wherein PCR amplification comprises 40 cycles, and the first five cycles are performed at a lower annealing/extension temperature.  
     
     
         27 . The method of  claim 26  wherein PCR amplification comprises 40 cycles, and the first two cycles are performed at a lower annealing/extension temperature.  
     
     
         28 . The method of  claim 25  wherein PCR amplification comprises 40 cycles, and the first two to five cycles are performed at a temperature of about 40° C. to 58° C.  
     
     
         29 . The method of  claim 28  wherein said early cycles are performed at a temperature of about 50° C.  
     
     
         30 . The method of  claim 15  further comprising the steps of: 
 (d) converting the cDNA obtained in step (b) to double-stranded DNA; and    (e) amplifying the RNA by subjecting the double-stranded DNA obtained in step (d) to in vitro transcription with an RNA polymerase to obtain amplified complementary RNA (cRNA).    
     
     
         31 . The method of  claim 30  wherein in step (d) the cDNA is converted to double-stranded DNA using RNaseH and DNA polymerase I.  
     
     
         32 . The method of  claim 30  wherein the amplified cRNA from step (e) is used directly as a template in a gene expression profiling assay by RT-PCR.  
     
     
         33 . The method of  claim 30  wherein step (e) includes labeling of the amplified cRNA with a detectable label.  
     
     
         34 . The method of  claim 33  wherein the detectable label is biotin or a fluorescent label.  
     
     
         35 . The method of  claim 30  comprising subjecting the amplified cRNA obtained in step (e) to hybridization to a microarray.  
     
     
         36 . The method of  claim 1  wherein the polyadenylated fragmented RNA obtained in step (a) is converted to cDNA by treatment with a reverse transcriptase and extended reverse primers, and the cDNA obtained is amplified by PCR using non-extended forward and a reverse PCR primers and probes, designed based on target amplicons.  
     
     
         37 . The method of  claim 36  wherein the extended reverse primers extend 10 bases further into the amplicon than the gene specific reverse PCR primer.  
     
     
         38 . The method of  claim 36  wherein the extended reverse primers extend 20 bases further into the amplicon than the gene specific reverse PCR primer.  
     
     
         39 . The method of  claim 36  wherein the extended reverse primers extend 30 bases further into the amplicon than the gene specific reverse PCR primer.  
     
     
         40 . The method of  claim 36  wherein the extended reverse primers extend into the amplicon within 1 base of the forward PCR primer.  
     
     
         41 . A method for enhanced cDNA synthesis comprising converting RNA to cDNA by treatment with a reverse transcriptase and extended primers, and amplifying the cDNA obtained by PCR using a forward and a reverse PCR primer and a probe, designed based on a target amplicon.  
     
     
         42 . The method of  claim 41  wherein said RNA is fragmented.  
     
     
         43 . The method of  claim 42  wherein at least part of said fragmented RNA is non-polyadenylated.  
     
     
         44 . The method of  claim 43  wherein the extended reverse primers extend 10 bases further into the amplicon than the reverse PCR primer (GSP).  
     
     
         45 . The method of  claim 43  wherein the extended reverse primers extend 20 bases further into the amplicon than the reverse PCR primer (GSP).  
     
     
         46 . The method of  claim 43  wherein the extended reverse primers extend 30 bases further into the amplicon than the reverse PCR primer (GSP).  
     
     
         47 . The method of  claim 43  wherein the extended reverse primers extend to within 1 base of the forward PCR primer.  
     
     
         48 . A method for preparing RNA comprising a multiplicity of RNA species for gene expression analysis comprising the steps of: 
 (a) polyadenylating said RNA; and    (b) converting the polyadenylated RNA to cDNA by reverse transcriptase and oligo dT or oligo dT-T7 primers.    
     
     
         49 . The method of  claim 48  wherein said RNA comprises RNA species with blocked 3′-termini.  
     
     
         50 . The method of  claim 49  comprising deblocking said blocked RNA species prior to step (a).  
     
     
         51 . The method of  claim 48  further comprising the step of using the oligo dT primed cDNA directly for gene expression analysis.  
     
     
         52 . The method of  claim 51  wherein said gene expression analysis is performed by TaqMan®.  
     
     
         53 . The method of  claim 48  further comprising the step of converting the oligo dT-T7 primed cDNA to double-stranded DNA with DNA polymerase I and RNAse H, and amplifying the double-stranded DNA with T7 RNA polymerase and rNTPs.  
     
     
         54 . The method of  claim 48  further comprising the step of converting the oligo dT-T7 primed cDNA to double-stranded DNA, and amplifying the double-stranded DNA with T7 RNA polymerase and biotinylated rNTPs.  
     
     
         55 . A method for preparing RNA comprising a multiplicity of RNA species for gene expression analysis comprising the steps of: 
 (a) polyadenylating said RNA; and    (b) converting the polyadenylated RNA to cDNA by reverse transcriptase and oligo dT-T7 primers containing a T7 RNA polymerase promoter, and    (c) subjecting the double-stranded DNA obtained in step (b) to in vitro transcription with a T7 RNA polymerase to obtain amplified complementary RNA (cRNA).