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-α real-time RT-PCR. The primers were desig...

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Bibliographic Details
Published in:Journal of virological methods 2006-03, Vol.132 (1), p.222-226
Main Authors: Tauriainen, Sisko, Dadu, Elina, Oikarinen, Maarit, Oikarinen, Sami, Hyöty, Heikki
Format: Article
Language:English
Subjects:
Biological and medical sciences
Control RNA
DNA Primers
Electrophoresis, Agar Gel
Enterovirus
Enterovirus - genetics
Fundamental and applied biological sciences. Psychology
GTP-Binding Proteins - genetics
Insulin - genetics
Interferon-alpha - genetics
Microbiology
NASBA
Reference Standards
Reverse Transcriptase Polymerase Chain Reaction - standards
RNA - biosynthesis
RNA amplification
RNA, Viral - analysis
RT-PCR
Self-Sustained Sequence Replication
Techniques used in virology
Virology
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Summary: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-α 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.
ISSN:0166-0934
1879-0984
DOI:10.1016/j.jviromet.2005.09.010