PolyA PCR Amplification of cDNA from RNA Extracted from Formalin-Fixed Paraffin-Embedded Tissue

Abstract

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.

Simultaneous Quantitative Detection of Relevant Biomarkers in Breast Cancer by Quantitative Real-Time PCR

“…However, most tumor samples are stored in paraffin blocks and the RNA in these tissues is strongly degraded (39), which may lead to a loss of detectable mRNA because RNA breaks can occur in between the location of the primers used. To set up a system which works with RNA from paraffin-embedded tissue it is necessary to either choose primer couples generating very small amplicons so that at least some of the extracted RNA can be amplified (40,41), or use newly developed protocols for fixation that prevent strong degradation (42).…”

Clinical quantitation of immune signature in follicular lymphoma by RT-PCR–based gene expression profiling

“…Total RNA was then extracted an RNeasy mini kit (Qiagen), as recommended by the manufacturer; DNase was used to remove the contaminating genomic DNA, and polyA RT-PCR was carried out as previously described. 13,[19][20][21] Global amplification of cDNA corresponding to all expressed genes (polyA PCR) was carried out as previously reported. [19][20][21] …”

“…12 The polyA cDNA pool generated is also indefinitely renewable and as such represents a "molecular block." [11][12][13] The polyA cDNA can be then be assayed for the expression of particular genes, either by hybridization with cDNA microarrays 14 or by real-time PCR. Real-time PCR measurement, however, enables more precise quantitation of the expression levels of specific Indicator genes.…”

A nondestructive molecule extraction method allowing morphological and molecular analyses using a single tissue section

“…In the early 1990s, several groups reported that DNA and RNA remained well preserved in FFPE and could be extracted for PCR amplification, although the mRNA size would be substantially reduced. [9][10][11][12] Only recently have researchers succeeded in developing protein extraction methods for FFPE tissues. 6,[13][14][15] However, these processes are destructive and require several hours, substantial amounts of tissues, and high salt concentrations in order to achieve satisfactory protein yields for SDS-PAGE analysis.…”