PMC:7068163 / 13088-14296
Annnotations
{"target":"https://pubannotation.org/docs/sourcedb/PMC/sourceid/7068163","sourcedb":"PMC","sourceid":"7068163","source_url":"https://www.ncbi.nlm.nih.gov/pmc/7068163","text":"Contamination as a reason for unspecific signals was ruled out, as stringent prevention measures were taken, e.g. strict separation of working areas: oligonucleotides and PCR mastermix reagents were handled in one room under a PCR hood with specified laboratory coats. Sample preparation and RNA extraction took place in a second room. Sample RNA was added in a third room under a PCR hood. The synthetic E gene control was added last to the mastermix. All reagents were aliquoted and aliquots used once only. Contaminations from synthetic E gene present in primer batches upon delivery can be ruled out as well, although only one batch of E gene primers and probes was used with the QuantiTect and Superscript III setup, as only a certain proportion of samples showed the unspecific signals. Furthermore, the unspecific signals were significantly reduced in the Superscript III setup, which showed that its sensitivity was comparable to the QuantiTect setup. In addition, the initially used E gene primers and probe were separately used as templates with the RealStar kit and no amplification was observed, whereas the corresponding artificial E gene template delivered a clear S-shaped curve with this 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