RT-PCR assays are unable to distinguish between RNA pol III transcribed Alu transcripts and RNA pol II transcribed mRNAs containing Alu sequences. A. Schematic of a bona fide pol III Alu transcript. Transcripts of 300 to 600 bp in length contain an Alu body (orange) flanked at the 3′ end by a poly-A stretch and the unique region (blue) which is determined by the location of the pol III terminator (Us). The poly-A stretch may be either homogeneous or heterogeneous containing non-adenosine bases. B. Schematic of an mRNA containing an Alu sequence. The Alu (orange) present within the mRNA (blue) will also have an oligo dA stretch at its 3′ end. Most standard RT-PCR approaches, such as 3′ RACE, rely on generating a cDNA through the reverse transcription of the RNA using an oligo dT primer (black arrow). Because both types of transcripts (pol II versus pol III) contain Alu sequences flanked by a polyA stretch, both will be amplified during reverse transcription. PCR amplification of selected cDNAs can then be performed by using a gene specific primer (in this case Alu, shown as an orange arrow) and a primer to the 3′ sequence of the oligo dT (represented as ‘Ns’). The PCR products (shown as black bars) of the cDNAs generated by both types of transcripts will yield the same type of product, thus making it difficult to distinguish the data generated from the bona fide pol III Alu transcripts.