Amplifying control RNA for RT-PCR applications by nucleic acid sequence based amplification (NASBA).
Control RNA for RT-PCR applications was amplified by nucleic acid sequence based amplification (NASBA) using the NucliSens Basic Kit. This method was used to construct positive control RNA for enterovirus, insulin, and G-protein RT-PCR, and for interferon-alpha real-time RT-PCR. The primers were designed to amplify identical RNA from RNA templates, which differs from the usual NASBA procedure, where opposite strand RNA is amplified from the target. This "inverse NASBA" method is easy to use and it does not require any expensive special equipment. The amplification reaction is done using a water bath and detection of amplified product by agarose gel electrophoresis. Generated RNA fragments were 195-714 bases long, of positive polarity and the amount of RNA was sufficient for thousands of RT-PCR reactions depending on the sensitivity of the RT-PCR.
Tauriainen
Sisko
S
Department of Virology, University of Tampere, Medical School, Biokatu 10, FIN-33520 Tampere, Finland. Sisko.Tauriainen@uta.fi
Dadu
Elina
E
Oikarinen
Maarit
M
Oikarinen
Sami
S
Hyöty
Heikki
H
D017931:DNA Primers; D004587:Electrophoresis, Agar Gel; D004770:Enterovirus / Q000235:genetics; D019204:GTP-Binding Proteins / Q000235:genetics; D007328:Insulin / Q000235:genetics; D016898:Interferon-alpha / Q000235:genetics; D012313:RNA / Q000096:biosynthesis*; D012367:RNA, Viral / Q000032:analysis; D012015:Reference Standards; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000592:standards*; D021161:Self-Sustained Sequence Replication
D017931:DNA Primers; D007328:Insulin; D016898:Interferon-alpha; D012367:RNA, Viral; D012313:RNA; D019204:GTP-Binding Proteins