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  • Secondary structure in the 3' UTR of EGF and the choice of reverse transcriptases affect the detection of message diversity by RT-PCR.

Secondary structure in the 3' UTR of EGF and the choice of reverse transcriptases affect the detection of message diversity by RT-PCR.

BioTechniques (1995-11-01)
E M Brooks, L G Sheflin, S W Spaulding
RESUMEN

The secondary structure in mRNA is essential for many processes, but it can present a technical problem in making full-length cDNA with reverse transcriptases. Furthermore, different reverse transcriptases have differing abilities to transcribe through regions with secondary structure, which can alter the products obtained by reverse-transcribing RNA and then PCR-amplifying the product (RT-PCR). We have been interested in studying the posttranscriptional regulation of epidermal growth factor by RT-PCR and have tested the ability of several reverse transcriptases to reverse transcribe the 3'-untranslated region (3'UTR), a region that contains substantial secondary structure. When low levels of either total RNA or poly(A)+ mRNA were used, we found avian myeloblastosis virus reverse transcriptase (AMV-RT) to be the most robust of all the enzymes tested. Furthermore, contrary to reports that AMV-RT is inhibited by tRNA--which should make it less effective than Moloney murine leukemia virus reverse transcriptase (MMLV-RT) at reverse-transcribing total RNA--adding tRNA to poly(A)+ RNA actually increased the amount of specific RT-PCR product obtained with AMV-RT while it decreased the amount of product and enhanced mispriming with MMLV-RT. We found that pre-incubation of the oligo(dT) primer with total RNA at elevated temperature prior to reverse transcription improved the efficiency of both native and modified MMLV-RTs. These findings support the concept that secondary structures in RNA differentially affect the abilities of different reverse transcriptases to detect transcript diversity and raise the possibility that such structures could affect quantitation using RT-PCR with internal mRNA standards.

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Sigma-Aldrich
Enhanced Avian First Strand Synthesis Kit, Components for cDNA synthesis with enhanced AMV reverse transcriptase
Sigma-Aldrich
Random Nonamers, for use as primers in cDNA synthesis, 50 μM in H2O
Sigma-Aldrich
Enhanced Avian Reverse Transcriptase [eAMV RT], For reverse transcription at higher temperatures & rare mRNAs
Sigma-Aldrich
Oligo(dT)23, Anchored, 70 μM in H2O