SECONDARY STRUCTURE IN THE 3'-UTR OF EGF AND THE CHOICE OF REVERSE TRANSCRIPTASES AFFECT THE DETECTION OF MESSAGE DIVERSITY BY RT-PCR

The secondary structure in mRNA is essential for many processes, but i t can present a technical problem in making full-length cDNA with reve rse transcriptases. Furthermore, different reverse transcriptases have differing abilities to transcribe through regions with secondary stru cture, which can alter the products obtained by reverse-transcribing R NA and then PCR-amplifying the product (RT-PCR). We have been interest ed in studying the posttranscriptional regulation of epidermal growth factor by RT-PCR and have rested the ability of several reverse transc riptases 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,,ve Sound avian myel oblastosis vints reverse transcriptase (AMV-RT) to be the most robust of all the enzymes tested. Furthermore, contrary to reports that AMV-R T is inhibited by tRNA - which should make it less effective than Molo ney murine leukemia vints reverse transcriptase (MMLV-RT) al reverse-t ranscribing total RNA - adding tRNA to poly(A)(+) RNA actually increas ed 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 a t elevated temperature prior to reverse transcription improved the eff iciency of both native and modified MMLV-RTs. These findings support t he concept that secondary structures in RNA differentially affect the abilities of different, reverse transcriptases to detect transcript di versity and mise the possibility that such structures could affect qua ntitation using RT-PCR with internal mRNA standards.