New methods as alternative or corrective measures for the pitfalls and artifacts of reverse transcription and polymerase chain reactions (RT-PCR) in cloning chimeric or antisense-accompanied RNA

Abstract

We established new methods for cloning cDNA ends that start with reverse transcription (RT) and soon proceed with the synthesis of the second cDNA strand, avoiding manipulations of fragile RNA. Our 3′-end cloning method does not involve poly-dT primers and polymerase chain reactions (PCR), is low in efficiency but high in fidelity and can clone those RNAs without a poly-A tail. We also established a cDNA protection assay to supersede RNA protection assay. The protected cDNA can be amplified, cloned and sequenced, enhancing sensitivity and fidelity. We report that RT product using gene-specific primer (GSP) cannot be gene- or strand-specific because RNA sample contains endogenous random primers (ERP). The gene-specificity may be improved by adding a linker sequence at the 5′-end of the GSP to prime RT and using the linker as a primer in the ensuing PCR. The strand-specificity may be improved by using strand-specific DNA oligos in our protection assay. The CDK4 mRNA and TSPAN31 mRNA are transcribed from the opposite DNA strands and overlap at their 3′ ends. Using this relationship as a model, we found that the overlapped sequence might serve as a primer with its antisense as the template to create a wrong-template extension in RT or PCR. We infer that two unrelated RNAs or cDNAs overlapping at the 5′- or 3′-end might create a spurious chimera in this way, and many chimeras with a homologous sequence may be such artifacts. The ERP and overlapping antisense together set complex pitfalls, which one should be aware of.

Cyclin-dependent kinase 4 may be expressed as multiple proteins and have functions that are independent of binding to CCND and RB and occur at the S and G2/M phases of the cell cycle

“…This question is of importance, because peers, when using RNAi technology, often select those shRNAs or siRNAs that work "fine" without asking why many others do not work. Over 63% of RNA transcripts, including the CCND1 mRNA, 48 from the human genome are accompanied by antisense counterparts, 49,50 and the Univergene database of the NCBI has over 123 000 human antisense entries. 51 Almost the whole human genome is transcribed, but all exons that encode proteins sum up to only 1.5% of the genomic sequence, with the hefty majority of the remaining transcripts mainly acting as regulatory RNAs.…”

“…The mRNAs of these 2 genes have a sense-antisense relationship and overlap at their last 517 nt, as we outlined recently. 48 A question thus arises as to how the cell knows whether it is the sense or the antisense RNA that should be knocked down when we force the cell to express a double-stranded regulatory RNA, such as an siRNA, shRNA, or microRNA. This question is of importance, because peers, when using RNAi technology, often select those shRNAs or siRNAs that work "fine" without asking why many others do not work.…”

Possible Formation of Mitochondrial-RNA Containing Chimeric or Trimeric RNA Implies a Post-Transcriptional and Post-Splicing Mechanism for RNA Fusion

“…This may partly be because a set of processed mtRNAs are not polyadenylated [17], leaving them a higher chance to form spurious chimeras as we inferred recently [15].…”

“…6). Because it was detected by RT-PCR that lacks the strand-specificity, as described by us recently [15], it is unclear whether this novel mtRNA is transcribed from the H- or the L-strand of the mtDNA.…”

“…4). Although most ESTs may be resulted from RT-PCR related techniques and thus are poor in the strand-specificity as we described recently [15], the interrelationship of the two mt-sequences within an EST should not be changed. The most convincing evidence for cis-splicing of mtRNA is our identification of the 16SrRNA-s in both HEK293 and Hela cells, as it is not associated with a deletion in the mtDNA (Fig.…”