Reverse transcription quantitative real-time PCR (RT-qPCR) is the most commonly used method for the quantification of mRNA expression levels.
Generally, the starting material for RTqPCR is purified RNA or mRNA. Since RNA does not serve as a template for PCR, it must first be copied into cDNA using a reverse transcriptase. The cDNA is then amplified by PCR to detectable levels for the quantification of gene expression.
RT-qPCR can be performed either as a coupled, one-step procedure (with RT and PCR performed in a single tube) or as a two-step procedure (with RT and PCR in separate tubes). This tutorial describes an accelerated workflow for two-step RT-qPCR procedures for gene-expression profiling of cultured cells (Figure 1).
Conventional column-based multi-step RNA purification methods deliver high-quality RNA, although the preparation methods are tedious and time-consuming. Therefore, new procedures that streamline and accelerate RNA preparation by fast and convenient cell lysis have become increasingly popular for gene-expression analysis.
Roche's RealTime ready Cell Lysis Kit, together with other Roche cDNA synthesis and real-time PCR products, provides an easy streamlined workflow for facilitating fast, reproducible gene-expression data.
Materials, Methods, and Results
The HepG2 cell line, derived from human liver carcinoma, was used for the research experimental setup. Prior to plating, cell number and cell viability were measured using the Cedex XS image-based automated cell culture analyzer.
HepG2 cells were seeded into E-Plates 96 at a density of 2.5 x 104 cells/well. Cells were plated in 200 µL MEM, supplemented with 10% fetal calf serum, 1% nonessential amino acids, L-glutamine (2 nM), penicillin (100 U/mL), and streptomycin (0.1 mg/mL), and cultured at +37eC in a 5% CO2-humidified atmosphere.
Cell growth behavior was continuously monitored using the xCELLigence RTCA MP instrument. Background impedance was measured in 100 µL cell culture medium per well. After plating, impedance was recorded at 15-minute intervals.
After 24 hours, the HepG2 cells were treated with different concentrations of doxorubicin (200 nM, 2 µM, 20 µM, and 200 µM), a drug used in chemotherapy. After compound administration, impedance was recorded in two-minute intervals for one hour followed by five-minute intervals for two hours, and fifteen-minute intervals for the remaining time.
Cell Index (CI) values were normalized to the time point of compound administration (referred to as normalized CI).
Two E-Plates 96 were used in parallel for the treatment approach: one E-Plate was continuously monitored for 96 hours; the second experiment was stopped eight hours post-treatment for cell lysis and gene-expression profiling.
After the completion of the experiment, the untreated (PBS control cells) and treated HepG2 cells were washed once within the EPlates (100 µL ice-cold PBS). Only treatment groups with a measured significant change in the proliferation rate (2 µM, 20 µM, and 200 µM) were profiled in the following RTqPCR experiments regarding expression of key genes of the cellular apoptotic pathway. All samples were run in technical triplicates through cDNA synthesis and qPCR analysis.
For fast RNA allocation, time-consuming and cumbersome conventional RNA-preparation methods were bypassed by using the RealTime Cell Lysis Kit (according to the package insert: 40 µL total lysis volume/reaction, 96-well format). This quick single-step cell lysis requires no intervention during its short incubation time.
The resulting lysate was directly reverse transcribed for fast cDNA synthesis (2 µL of the total lysate/cDNA reaction) using Transcriptor Universal cDNA Master according to the package insert (20 µL total cDNA reaction volume).
Optionally, DNase treatment can be integrated into the cDNA synthesis protocol. This combination of cDNA synthesis and DNase treatment streamlines the entire experimental workflow and reduces the overall number of single steps, thus increasing convenience together with minimizing error sources and sample loss (total reaction time 25 minutes).
An additional benefit provided by the Real-Time ready Cell Lysis Kit is the usage of a thermolabile DNase that eliminates the need for an extra heating step required by conventional approaches using thermostable enzymes.
Overall, the use of the RealTime ready Cell Lysis Kit accelerates the entire experimental setup of gene-expression studies by reducing handling steps and incubation times, and eliminating the need for user intervention (Figure 2). Therefore, this accelerated workflow can be integrated into any automated medium- to high-throughput workflow.
qPCR and Data Analysis
qPCR was performed on the LightCycler® 480 Real-Time PCR Instrument (96 well) using the RealTime ready Human Apoptosis Panel 96 in combination with the LightCycler® 480 Probes Master (96-well format: 5 µL cDNA, 15 µL PCR mix, standard PCR protocol).
The RealTime ready Human Apoptosis Panel 96 is part of the family of ready-to-use Focus Panels, which provide preplated target gene sequences for the gene-expression profiling of dedicated focus areas.
The RealTime ready Cell Lysis Kit represents a beneficial building block in the pathway of gene-expression analysis by providing significant time savings combined with increased convenience for low- to mediumthroughput, real-time PCR applications.
Together with the Transcriptor Universal cDNA Master, with its two-component kit concept and function-tested RealTime ready assays, even complex gene-expression studies can be made easier and more effective, providing savings in time and resources. By supporting high-quality results in target profiling, these products are an attractive solution for meeting diverse research challenges, whether one is studying a few cells or thousands of cells.