tetano
Editor, Senior Moderator
Sci Rep. 2014 Apr 11;4:4659. doi: 10.1038/srep04659.
A simple, inexpensive method for preparing cell lysates suitable for downstream reverse transcription quantitative PCR.
Shatzkes K1, Teferedegne B2, Murata H3.
Author information
Abstract
Sample nucleic acid purification can often be rate-limiting for conventional quantitative PCR (qPCR) workflows. We recently developed high-throughput virus microneutralization assays using an endpoint assessment approach based on reverse transcription qPCR (RT-qPCR). The need for cumbersome RNA purification is circumvented in our assays by making use of a commercial reagent that can easily generate crude cell lysates amenable to direct analysis by one-step RT-qPCR. In the present study, we demonstrate that a simple buffer containing a non-ionic detergent can serve as an inexpensive alternative to commercially available reagents for the purpose of generating RT-qPCR-ready cell lysates from MDCK cells infected with influenza virus. We have found that addition of exogenous RNase inhibitor as a buffer component is not essential in order to maintain RNA integrity, even following stress at 37?C incubation for 1-2 hours, in cell-lysate samples either freshly prepared or previously stored frozen at -80?C.
PMID:
24722424
[PubMed - in process]
http://www.ncbi.nlm.nih.gov/pubmed/24722424
A simple, inexpensive method for preparing cell lysates suitable for downstream reverse transcription quantitative PCR.
Shatzkes K1, Teferedegne B2, Murata H3.
Author information
Abstract
Sample nucleic acid purification can often be rate-limiting for conventional quantitative PCR (qPCR) workflows. We recently developed high-throughput virus microneutralization assays using an endpoint assessment approach based on reverse transcription qPCR (RT-qPCR). The need for cumbersome RNA purification is circumvented in our assays by making use of a commercial reagent that can easily generate crude cell lysates amenable to direct analysis by one-step RT-qPCR. In the present study, we demonstrate that a simple buffer containing a non-ionic detergent can serve as an inexpensive alternative to commercially available reagents for the purpose of generating RT-qPCR-ready cell lysates from MDCK cells infected with influenza virus. We have found that addition of exogenous RNase inhibitor as a buffer component is not essential in order to maintain RNA integrity, even following stress at 37?C incubation for 1-2 hours, in cell-lysate samples either freshly prepared or previously stored frozen at -80?C.
PMID:
24722424
[PubMed - in process]
http://www.ncbi.nlm.nih.gov/pubmed/24722424