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A simple, inexpensive method for preparing cell lysates suitable for downstream reverse transcription quantitative
Kenneth Shatzkes1, Belete Teferedegne2, Haruhiko Murata3
11] Laboratory of DNA Viruses, Division of Viral Products, OVRR, CBER, FDA, Bethesda, MD 20892, USA [2] Laboratory of Pediatric and Respiratory Viral Diseases, Division of Viral Products, OVRR, CBER, FDA, Bethesda, MD 20892, USA [3] [4].
A simple detergent buffer can replace expensive reagents for preparing cell lysates for quantitative reverse transcription PCR (RT-qPCR). This method simplifies influenza virus detection and RNA analysis without compromising RNA integrity.
Area of Science:
- Molecular Biology
- Virology
- Biochemistry
Background:
- Nucleic acid purification is a bottleneck in quantitative PCR (qPCR) workflows.
- High-throughput virus microneutralization assays utilize reverse transcription qPCR (RT-qPCR) with endpoint assessment.
- Commercial reagents facilitate direct analysis of crude cell lysates by RT-qPCR, bypassing RNA purification.
Purpose of the Study:
- To evaluate an inexpensive non-ionic detergent buffer as an alternative to commercial reagents for generating RT-qPCR-ready cell lysates.
- To assess the necessity of exogenous RNase inhibitor for maintaining RNA integrity in cell lysates.
Main Methods:
- Preparation of cell lysates from influenza virus-infected MDCK cells using a simple detergent buffer.
- Analysis of cell lysates by one-step RT-qPCR.
- Assessment of RNA integrity in freshly prepared and frozen cell lysates subjected to thermal stress.
Main Results:
- A simple non-ionic detergent buffer effectively generates RT-qPCR-ready cell lysates.
- Exogenous RNase inhibitor is not essential for maintaining RNA integrity in cell lysates.
- RNA integrity is preserved even after incubation at 37 °C for 1-2 hours in both fresh and frozen samples.
Conclusions:
- An inexpensive detergent buffer offers a cost-effective alternative for cell lysate preparation in RT-qPCR assays.
- The developed method simplifies high-throughput virus microneutralization assays.
- This approach streamlines influenza virus detection and RNA analysis without compromising sample quality.
