Sidebar: Guidelines for qPCR Consistency
The exceptional success and widespread use of qPCR has led to a prodigious number of publications (~18,000 in 2008). Despite its popularity, the technology lacks a consensus on how best to perform and interpret results. A consortium of experts in the field recently provided new guidelines to help deal with this challenge.
The MIQE Guidelines provide the minimum information for publication of quantitative real-time PCR experiments. They cover a host of recommendations such as the need to provide assay details about reagent components, oligonucleotide sequences, assay sensitivity, and efficiency.
According to co-author Tania Nolan, Ph.D., global manager, technical and application support team, Sigma-Aldrich, “Over time scientists have adopted many different protocols and these differences lead to variations in data. It is impossible to interpret scientific findings unless the relevant methods have been clearly explained. The guidelines are directed to anyone performing qPCR and also to those editing peer-reviewed articles where qPCR data is included. In addition, the guidelines have been sent to journal editors and are being adopted by journals.”
Although they are voluntary, the response has been positive, reports Dr. Nolan. “Journals are considering how they can implement them, and companies are responding by addressing how they can enable scientists to include the required information. Most scientists welcome this greater transparency of scientific reports.”
Sidebar: New Twist on RT-qPCR Sample Prep
Conventional methods for RNA purification typically involve time-consuming steps and require 30 minutes or more to complete. According to Viresh Patel, Ph.D., senior product manager, PCR reagents, Bio-Rad Laboratories, these approaches usually incorporate one or more of the following techniques—organic extraction, magnetic particles, or salting out—and do not always result in purified RNA that is free of contaminating genomic DNA.
“This is particularly problematic for organic extraction of nucleic acids, which requires a DNase step to remove carryover DNA, followed by exposure to heat (~75°C for 5–10 minutes) to inactivate the DNase before proceeding,” explains Dr. Patel.
“Heating RNA has been shown to result in degradation, especially in the presence of Mg++. Column-based methods that include salting out or magnetic particles provide more efficient RNA purification, though researchers still rely on the use of DNases to ensure RNA purity.”
Using Cell Lysates
Late last month Bio-Rad introduced its iScript™ RT-qPCR sample-preparation reagent for isolation of total RNA and for enabling reverse transcription and real-time PCR to be performed directly from cell lysates. Scientists can use the protocol to remove genomic DNA and stabilize RNA in as little as five minutes, claims Dr. Patel.
“We designed [the product] to meet the needs of researchers performing high-throughput gene-expression analysis, gene silencing, and microarray validation,” he says.
Upon exposure of cells to the sample-prep reagent, cell disruption, and lysis occur in approximately 30 seconds with the aid of mild vortexing, notes Dr. Patel in describing the process.
“The reagent also stabilizes RNA and inhibits intracellular ribonucleases. A short, i.e., two minute, high-speed centrifugation yields total RNA that can be collected in the supernatant, while the nuclei containing genomic DNA form a pellet and can be discarded,” he continues, adding that the resulting RNA preparation is ready for use, without further treatment, in subsequent reverse transcription and PCR/qPCR.