A simple, inexpensive method for preparing cell lysates suitable for downstream reverse transcription quantitative PCR

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.

Encoded Hydrogel Microparticles for Sensitive and Multiplex microRNA Detection Directly from Raw Cell Lysates

“…Generally, it has been shown that miRNAs are highly stable in cell lysates and can be stored at −20 and/or −80°C for days. 18,19 In our proof-ofconcept test of freeze−thaw cell lysate, the endogenous miR-21 signal was decreased by ∼50% compared to fresh cell lysates ( Figure S4). This signal decrease is probably due to freeze/ thaw temperatures (here, −20°C/room temperature) or absence of RNase inhibitor; a previous study demonstrated that RNA stability was enhanced when stored at −80°C or thawed at 4°C or RNase inhibitor added.…”

“…Previously, Ho et al has reported microRNA LODs of 200 cells, 19 which is within an order of magnitude of the LOD obtained with our hydrogel-based detection. Even though some recent studies on direct miRNA measurements demonstrate high sensitivity (1−1000 cells), 18,20 their measurements are based on target-based amplification of qRT-PCR. This targetbased amplification often introduces sequence bias.…”

“…This signal decrease is probably due to freeze/ thaw temperatures (here, −20°C/room temperature) or absence of RNase inhibitor; a previous study demonstrated that RNA stability was enhanced when stored at −80°C or thawed at 4°C or RNase inhibitor added. 18 For better assay reliability and sensitivity, it is possible to further optimize our assay protocol in the future.…”

Histone Acetyltransferase (HAT) P300/CBP Inhibitors Induce Synthetic Lethality in PTEN-Deficient Colorectal Cancer Cells through Destabilizing AKT

“…Cells were washed with cold PBS once and lysed in the Real-time cell lysis buffer containing 10mM Tris pH 7.4, 0.25% Igepal CA-630, and 150mM NaCl for 5 mins [18]. Cell lysates were directly used for real-time as the amplification templates.…”

Activation of Yap-Directed Transcription by Knockdown of Conserved Cellular Functions

“…Cell lysate was then prepared by aspirating the culture media using the GNF Systems (San Diego, CA) WDII plate washer, and then the cells were washed with 100 μL of cold PBS, removing as much of the wash solution from each well as possible. The cells were then lysed by the addition of 15 μL of lysis buffer (10 mM Tris-HCl, pH 7.4, 0.25% IGEPAL, 150 mM NaCl, final concentrations, dissolved using DNase/ RNase-free water, all from Sigma) as described by Shatzkes et al 20 The cells were lysed by incubation for 30 min at room temperature. During this time, the PCR master mix (Roche Diagnostics, Indianapolis, IN) was prepared, including Taqman primers for actin and CYR61, and then 900 nL of the master mix was dispensed to the Roche LightCycler 1536 well assay plate using the Thermo Combi nL.…”