Are there specific recommendations for performing RT-PCR on RNA isolated from paraffin-embedded samples?
Paraffin-embedded or fixed samples typically yield fragmented, partially degraded RNA. In addition, RNA quality will depend greatly on the handling of the samples before, during, and after the fixation procedure.
If performing RT-PCR with degraded RNA, we recommend for cDNA synthesis the use of gene-specific primers or random nonamers rather than oligo-dT primers, since the mRNA poly-A tail may have been lost due to degradation. Regarding PCR primer design, shorter amplicon sizes are preferable.
If performing RT-PCR with degraded RNA, we recommend for cDNA synthesis the use of gene-specific primers or random nonamers rather than oligo-dT primers, since the mRNA poly-A tail may have been lost due to degradation. Regarding PCR primer design, shorter amplicon sizes are preferable.