title: Depletion of polycistronic transcripts using short interfering RNAs: cDNA synthesis method affects levels of non-targeted genes determined by quantitative PCR.
abstract: BACKGROUND
  Short interfering RNAs (siRNAs) are often used to deplete viral polycistronic transcripts, such as those encoded by human papillomavirus (HPV). There are conflicting data in the literature concerning how siRNAs targeting one HPV gene can affect levels of other genes in the polycistronic transcripts. We hypothesised that the conflict might be partly explained by the method of cDNA synthesis used prior to transcript quantification.
  
  
  RESULTS
  We treated HPV16-positive cervical keratinocytes with siRNAs targeting the HPV16 E7 gene and used quantitative PCR to compare transcript levels of E7 with those of E6 and E2, viral genes located upstream and downstream of the target site respectively. We compared our findings from cDNA generated using oligo-dT primers alone with those from cDNA generated using a combination of random hexamer and oligo-dT primers. Our data show that when polycistronic transcripts are targeted by siRNAs, there is a period when untranslatable cleaved mRNA upstream of the siRNA binding site remains detectable by PCR, if cDNA is generated using random hexamer primers. Such false indications of mRNA abundance are avoided using oligo-dT primers. The period corresponds to the time taken for siRNA activity and degradation of the cleaved transcripts. Genes downstream of the siRNA binding site are detectable during this interval, regardless of how the cDNA is generated.
  
  
  CONCLUSIONS
  These data emphasise the importance of the cDNA synthesis method used when measuring transcript abundance following siRNA depletion of polycistronic transcripts. They provide a partial explanation for erroneous reports suggesting that siRNAs targeting HPV E7 can have gene-specific effects.
authors.affiliation: Department of Pathology, University of Cambridge, Tennis Court Road, Cambridge CB2 1QP, UK.
authors.forename: Jennifer E
authors.initials: JE
authors.lastname: Hanning
authors.forename: Ian J
authors.initials: IJ
authors.lastname: Groves
authors.forename: Mark R
authors.initials: MR
authors.lastname: Pett
authors.forename: Nicholas
authors.initials: N
authors.lastname: Coleman
mesh_terms: D018076:DNA, Complementary / Q000096:biosynthesis; D005260:Female; D020869:Gene Expression Profiling / Q000379:methods*; D015967:Gene Expression Regulation, Viral; D052162:Human papillomavirus 16 / Q000235:genetics*; D006801:Humans; D015603:Keratinocytes / Q000821:virology; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism*; D034741:RNA, Small Interfering / Q000235:genetics / Q000378:metabolism*; D012367:RNA, Viral / Q000235:genetics / Q000378:metabolism*
chemical_list: D018076:DNA, Complementary; D012333:RNA, Messenger; D034741:RNA, Small Interfering; D012367:RNA, Viral