Scaling up in vitro transcription synthesis of RNA standards for competitive quantitative RT-PCR: Looking for bigger yields

Generation of HIV-1 and Internal Control Transcripts as Standards for an In-House Quantitative Competitive RT-PCR Assay to Determine HIV-1 Viral Load

“…The RNA concentration estimated for the transcript T-HIV1-2 b was similar to the ones obtained by González et al, in which the log 10 orders of estimated HCV RNA concentrations were higher than 11 for all transcripts they obtained [19]. In our study, we obtained HIV-1 RNA concentrations about 10 14  IU/mL (Table 5), allowing us to use the HIV-1 transcript as standard for quantification purposes in calibration curves that commonly have maximal concentrations with log 10 orders between 5 and 9 [19, 29].…”

“…In order to develop an in-house qRT-PCR assay for HIV-1 RNA and taking into account the very high yields that are obtained in the in vitro transcription reactions [19], we used the dilutions of the transcript T-HIV1-2 b in which DNA signal was not obtained. …”

“…In our study, we obtained HIV-1 RNA concentrations about 10 14  IU/mL (Table 5), allowing us to use the HIV-1 transcript as standard for quantification purposes in calibration curves that commonly have maximal concentrations with log 10 orders between 5 and 9 [19, 29]. …”

“…The correspondent HIV-1 and IC RNAs were obtained from 1  μ g of pGEM-HIV1 and pGEM-IC plasmids using 50 u of T7 RNA polymerase (Promega, USA) in presence of 0.75 mM of each rNTPs (Promega, USA), transcription buffer (Promega, USA) in a 50  μ L reaction, according to the procedure optimized in our laboratory, described in [19]. After transcription, 2 or 4 u of RNase free DNase (Promega, USA) per 1  μ g of template DNA, were added for 15 or 30 min at 37°C to degrade the contaminant DNA.…”