ArticleRNA biology2013
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.
Article in RNA biology, 2013. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 16 papers.
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Who cites it
16 citing papers in PubMed.
- Updating mRNA variants of the human RSK4 gene and their expression in different stressed situations.Heliyon · 2024Article
- Experimental and analytical pipeline for sub-genomic RNA landscape of coronavirus by Nanopore sequencer.Microbiology spectrum · 2024Article
- Chimeric RNAs Discovered by RNA Sequencing and Their Roles in Cancer and Rare Genetic Diseases.Genes · 2022Review
- RNA expression profiling from the liquid fraction of synovial fluid in knee joint osteoarthritis patients.American journal of translational research · 2022Article
- Pan-Cancer Analysis Reveals the Diverse Landscape of Novel Sense and Antisense Fusion Transcripts.Molecular therapy. Nucleic acids · 2020Article
- Long-read sequencing uncovers a complex transcriptome topology in varicella zoster virus.BMC genomics · 2018Article
- Review
- While it is not deliberate, much of today's biomedical research contains logical and technical flaws, showing a need for corrective action.International journal of medical sciences · 2018Review
- It Is Imperative to Establish a Pellucid Definition of Chimeric RNA and to Clear Up a Lot of Confusion in the Relevant Research.International journal of molecular sciences · 2017Review
- Evolutionary Insights into RNA trans-Splicing in Vertebrates.Genome biology and evolution · 2016Review
- Article
- An Xist-activating antisense RNA required for X-chromosome inactivation.Nature communications · 2015Article
- To Know How a Gene Works, We Need to Redefine It First but then, More Importantly, to Let the Cell Itself Decide How to Transcribe and Process Its RNAs.International journal of biological sciences · 2015Review
- Hypothesis: Artifacts, Including Spurious Chimeric RNAs with a Short Homologous Sequence, Caused by Consecutive Reverse Transcriptions and Endogenous Random Primers.Journal of Cancer · 2015Review
- 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 G 2/M phases of the cell cycle.Cell cycle (Georgetown, Tex.) · 2013Article
- Article
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Authors and funding
4 authors.
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No grant is acknowledged in the PubMed record.
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.
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