Amplifying control RNA for RT-PCR applications by nucleic acid sequence based amplification (NASBA).

Area of Science:

• Molecular Biology
• Virology
• Biotechnology

Background:

• Reverse transcription polymerase chain reaction (RT-PCR) is a vital technique in molecular biology.
• The availability of reliable positive control RNA is crucial for validating RT-PCR assays.
• Conventional NASBA amplifies the opposite strand RNA, necessitating specific primer design.

Purpose of the Study:

• To develop an efficient and accessible method for generating positive control RNA for various RT-PCR applications.
• To adapt the nucleic acid sequence based amplification (NASBA) technique for producing identical RNA strands.
• To establish a cost-effective alternative for producing RNA controls for molecular diagnostics.

Main Methods:

• Utilized the NucliSens Basic Kit for nucleic acid sequence based amplification (NASBA).
• Developed an "inverse NASBA" protocol where primers amplify identical RNA from RNA templates, unlike standard NASBA.
• RNA amplification was performed in a water bath, with product detection via agarose gel electrophoresis.

Main Results:

• Successfully generated positive control RNA for enterovirus, insulin, G-protein, and interferon-alpha RT-PCR.
• Produced RNA fragments of positive polarity, ranging from 195 to 714 bases in length.
• The generated RNA amount was sufficient for thousands of RT-PCR reactions, contingent on assay sensitivity.

Conclusions:

• The "inverse NASBA" method provides an easy-to-use and cost-effective way to produce positive control RNA.
• This technique eliminates the need for expensive specialized equipment, making it broadly applicable.
• The generated RNA controls are suitable for a wide range of RT-PCR and real-time RT-PCR applications.