PolyA PCR amplification of cDNA from RNA extracted from formalin-fixed paraffin-embedded tissue.
RNA extraction still relies almost exclusively on the use of fresh or frozen tissue, limiting the number of samples that can be analyzed, and there is a growing need for means of global mRNA analysis of archived formalin-fixed paraffin-embedded tissue (FFPET). Previous reports of RNA extraction and amplification from FFPET are limited and do not enable global cDNA amplification. This study used polyA PCR to generate globally amplified cDNA from RNA extracted from formalin-fixed paraffin-embedded samples. RNA was extracted from nine routinely processed archival FFPET samples (lymph node, nasopharynx, prostate, lung and bone marrow) using an Ambion Paraffin Block RNA Isolation Kit. Global cDNA was generated by polyA RT-PCR and used in GAPDH specific PCR and PCR for CD33, c-myb, and SNF2. PolyA cDNA was reamplified by polyA PCR and the reamplified cDNA also used in GAPDH PCR. RNA was extracted from all nine samples, but was degraded. PolyA RT-PCR generated cDNA from all samples and was positive for GAPDH PCR in seven. PCR for CD33, c-myb, and SNF2 was positive in all samples tested. Following reamplification, the polyA cDNA remained positive for GAPDH by PCR. The results demonstrate the feasibility of globally amplifying RNA isolated from archival FFPET samples using polyA RT-PCR, which generates a renewable cDNA pool that can be probed for any cDNA species and reamplified as necessary.
Byers
Richard
R
Laboratory Medicine Academic Group, Faculty of Medicine, Dentistry, Nursing, and Pharmacy, University of Manchester, England. r.byers@man.ac.uk
Roebuck
Jamie
J
Sakhinia
Ebrahim
E
Hoyland
Judith
J
D000241:Adenosine; D000293:Adolescent; D000328:Adult; D000368:Aged; D001483:Base Sequence; D017931:DNA Primers; D018076:DNA, Complementary / Q000138:chemical synthesis*; D005260:Female; D005557:Formaldehyde; D006801:Humans; D008297:Male; D008875:Middle Aged; D008969:Molecular Sequence Data; D016612:Paraffin Embedding; D011108:Polymers; D012313:RNA / Q000302:isolation & purification*; D020133:Reverse Transcriptase Polymerase Chain Reaction
D017931:DNA Primers; D018076:DNA, Complementary; D011108:Polymers; D005557:Formaldehyde; C069424:polyadenosine; D012313:RNA; D000241:Adenosine