Figure 6. Depiction of artifacts caused by ERP or by 5′ or 3′ overlapping. (A) Because the RNA sample contains ERP, RT with Linker-GSP will also generate the first cDNA strand of the antisense RNA, besides the cDNA of the desired sense RNA. When the RT product (usually only 1 µl) is added into the PCR mixture as the template, some Linker-GSP residual is transferred together, which primes the synthesis of a linker containing antisense fragment. The fragment is amplified in the later PCR cycles. However, because the PCR mixture contains many more copies of the GSP and the gene-specific reverse primer (GSRP) than the Linker-GSP residual, the first PCR cycle should generate many more copies of the desired sense cDNA, which titrates out the antisense in later PCR cycles, unless the antisense RNA is expressed at a much higher level than the sense. We use as small an amount of linker-GSP as possible in the RT to minimize its residual in the RT product. (B) Two cDNAs that overlap at their 5′ ends, no matter whether they are unrelated or are originated respectively from a sense and an antisense transcripts that overlap at their 3′ ends (like CDK4 and TSPAN31), can be converted to 3′-overlapped counterparts after one round of PCR by GSP or ERP, which, in turn, creates wrong-template extension in later PCR cycles as depicted in Figure 5C. (C) If a cDNA has an unprotected 3′ end that is reverse-complementary to an unrelated cDNA (in red color), this matched part (e.g., ATCGA/TAGCT) and this other cDNA may serve in PCR as the primer and the template, respectively, to create a spurious chimera.