Template switching within exons 3 and 4 of KV11.1 (HERG) gives rise to a 5′ truncated cDNA

Transcriptional-Readthrough RNAs Reflect the Phenomenon of “A Gene Contains Gene(s)” or “Gene(s) within a Gene” in the Human Genome, and Thus Are Not Chimeric RNAs

“…On the other hand, more publications continue emerging to report [ 95 ] or summarize [ 3 , 69 , 70 ] such trans -splicing related chimeras or other noncolinear RNAs. Moreover, many bioinformatic experts are establishing different algorithms to cull chimeras from different sets of high-throughput sequencing data [ 96 , 97 , 98 , 99 , 100 , 101 , 102 , 103 , 104 ], although all these data sets contain many spurious sequences, as we and others have pointed out [ 5 , 6 , 64 , 105 , 106 , 107 , 108 , 109 , 110 , 111 , 112 , 113 , 114 , 115 , 116 , 117 , 118 , 119 , 120 , 121 , 122 , 123 , 124 , 125 , 126 , 127 , 128 , 129 , 130 , 131 , 132 , 133 , 134 , 135 ]. This situation is worrisome to us.…”

“…For example, splicing is initiated and finished too quickly to study its detail, as aforementioned. In addition, the reported detection of the abovementioned RNAs all involved reverse transcription (RT) and polymerase chain reactions (PCR), which are techniques that easily create spurious results, as we and others have repeatedly described before, due to template-switching, mis-priming, self-priming, DNA or complementary DNA (cDNA) damage, and PCR-reconditioning, among other reasons [ 5 , 6 , 64 , 105 , 106 , 107 , 108 , 109 , 110 , 111 , 112 , 113 , 114 , 115 , 116 , 117 , 118 , 119 , 120 , 121 , 122 , 123 , 124 , 125 , 126 , 127 , 128 , 129 , 130 , 131 , 132 , 133 , 134 , 135 ]. Therefore, approaches without involvement of RT and PCR are needed to minimize technical artifacts for indisputable evidence and to obtain procedural and mechanistic details of the presumed trans -splicing.…”

Hypothesis: Artifacts, Including Spurious Chimeric RNAs with a Short Homologous Sequence, Caused by Consecutive Reverse Transcriptions and Endogenous Random Primers

“…The second cDNA strand is then synthesized and PCR ensues to amplify the cDNA library 112 , which may or may not be followed by ligation to a vector or specific sequencing primers (depending on whether the prior primers contain an adaptor or not). During these RT and PCR procedures, artificial chimeric cDNAs may be formed 66 , 115 - 122 , in part because template switching may occur to skip the region in a secondary structure during RT 102 , 115 , 116 , 123 , 124 and mis-priming can occur in PCR, as having been well discussed in the literature 116 , 117 , 125 - 128 .…”

A PCR-based method for detection and quantification of small RNAs

“…These data suggested that the pre-miRNAs were eliminated during the srcDNA synthesis and that the srcDNAs contained only the mature miRNAs. In fact, all pre-miRNAs form a hairpin-loop in their secondary structure and the cDNA synthesis can be prematurely stopped or skipped on these hairpin loops during the reverse transcription (RT) reaction [20][21][22][23]. To overcome this problem, RT reactions should be performed at higher temperatures (60-70°C) using a high-temperature-stable Reverse Transcriptase [20,22,23].…”

“…In fact, all pre-miRNAs form a hairpin-loop in their secondary structure and the cDNA synthesis can be prematurely stopped or skipped on these hairpin loops during the reverse transcription (RT) reaction [20][21][22][23]. To overcome this problem, RT reactions should be performed at higher temperatures (60-70°C) using a high-temperature-stable Reverse Transcriptase [20,22,23]. Indeed, a recent report on quantitative analyses of pre-miRNA expression in cancer cell lines employed a high-temperature-stable Thermoscript to synthesize cDNAs at 60°C [24].…”