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J Biomol Tech. 2010 September; 21(3 Suppl): S44.
PMCID: PMC2918162

Novel Primer Design for cDNA Synthesis and qPCR: TR Priming with RNA Templates of Divergent Quality (RIN 9.5 to 2.2) Outperforms Oligo-dT Priming as well as Random Priming

S. Quabius2
1AmpTec GmbH, Hamburg, Germany;
2Institute for Immunology, UKSH, Kiel, Germany

Abstract

RP-75

A frequent problem in gene expression analysis is variable RNA quality, especially with clinical RNA samples. Oligo-dT primers provide cDNA from regions next to the 3'-poly-A tail. Internal transcript regions are (partially) lost and effects on different mRNAs and PCR amplicons are variable. Accordingly, gene expression results (RT-qPCR) vary widely with different RNA qualities. For example, upon RNA degradation, the Ct for actin mRNA increases 9.9 cycles, with much lower effects on other mRNAs, e.g. an increase of only 3.9 for YMHAZ. Although there is good selectivity against rRNA sequences in intact RNA: Ct for 18SrRNA is only -4.1 vs actin mRNA (17-fold more rRNA). Since rRNA is more stable, the difference increases to 9.0 in degraded RNA (500-fold more rRNA). With random primers (or a mix of random and oligo-dT), the recovery of internal transcript regions is improved. Accordingly, there is less variability with different RNA qualities: Ct for beta-actin increases by 5.4 cycles, comparable to 4.7 for YMHAZ. Selectivity against rRNA sequences is poor. Intact RNA: Ct for 18SrRNA vs actin is -9.2(588-fold more rRNA), with further increase to -10.7 (1663-fold more rRNA). With our novel TR primers, preferential priming occurs at the 3”;-end of RNA fragments (independent from poly-A) and different to random priming, further “cutting” of RNA fragments into multiple, short cDNAs is prevented; combined with improved recovery of internal transcript regions. Ct for beta-actin increases by 4.1, very comparable to 4.4 for YMHAZ. Furthermore, good selectivity against rRNA sequences. Intact RNA: Ct for 18SrRNA vs actin is -7.5 (181-fold more rRNA), a small increase to -8.6 (388-fold more rRNA, lower than for oligo-dT with this RNA sample). In addition to improved performance in RT-qPCR assays, this selectivity against rRNA sequences makes TR primers an ideal tool for the novel “Exon Microarrays”.


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