Quantitative polymerase chain reaction (qPCR) is one of the most powerful and sensitive means to measure gene expression. The ability to generate data in real time revolutionized the way PCR quantifies DNA or RNA.
Inconsistency of methodologies continues to plague the field, although the MIQE guidelines have helped by defining the minimum information needed to accurately evaluate qPCR. Nonetheless, this bustling field continues to improve and make steady progress. At BioEPS’ recent “QPCR” conference in Germany, advances in assay design and data interpretation were highlighted, and new applications ranging from cancer diagnostics to genotyping featured.
Real-time qPCR is often combined with reverse transcription to quantify cellular messenger or noncoding RNA. But it can also quantify a targeted DNA, an emerging application for the new field of epigenetics.
?“Assays for DNA methylation and histone modifications typically are used to assess epigenetic processes that control gene expression,” explained Viresh Patel, Ph.D., marketing manager, gene expression division, Bio-Rad. “This regulation is mediated through chromatin state changes. Actively transcribed genes have open or accessible, chromatin regions, while genes that are silent are in closed, or inaccessible, chromatin.”
Bio-Rad recently introduced its new EpiQ™ chromatin analysis kit, a tool for researchers who want to assess epigenetic regulation of genes using cultured cells. Dr. Patel said the kit is fast and easy.
“There are two challenges facing epigenetic researchers: time to results and the large amount of input sample needed for analysis. We developed this kit as a new type of assay that interrogates the functional state of chromatin inside the cell. It is easy to perform, quantitative, and produces results on the day of cell harvest. Basically, one lyses cells (as few as 50,000 cells), digests the chromatin in situ, purifies and then quantifies the DNA, and performs the qPCR reaction with specific primers.”
According to Francisco Bizouarn, international field application specialist, the company validated its assay by analyzing 15 genes in 4 human cancer cell lines in which they assessed 4 housekeeping genes and 11 genes that are epigenetically regulated.
“Our data discovered that contrary to the popular notion that gene promoters exist entirely in only open or closed chromatin states, there are intermediate states of chromatin accessibility, too. So there is a continuum from 0 to 100%.”
The EpiQ chromatin assays will be especially useful to complement existing datasets such as for typical assays for DNA methylation and histone modification. “The assays are also very useful for a scientist who wants to determine if epigenetics might be involved in the regulation of a specific target. All one needs is a primer set for target genes of interest and the kit.”
If you are not already testing for inhibitors in your PCR reaction, this may be the reason you see variable results,” warned Melissa Kelley, Ph.D., research scientist, Thermo Fisher Scientific. “One problem is that routine methods such as spectrophotometric determination of RNA quality can’t rigorously detect the presence of inhibitors such as EDTA, phenol, heparin, and ethanol. A second problem is the common misconception that inhibitors affect different gene targets to the same degree. Both of these issues can produce hidden sources of errors and cause inaccurate or incorrect data.”
The company has designed its Solaris RNA Spike Control Kit using an exogenous RNA control to identify common inhibitors. “A common technique used by researchers is the serial dilution method. But this requires more sample template and more wells—about 15–18 additional wells per assay. There are other kits currently available that use RNA or DNA spike-in molecules. But these may be less sensitive to inhibition because they are added at later steps in the workflow. We performed many optimization studies to determine where to add in a spike control and found that it was best to add it directly to the RNA sample and then do the cDNA synthesis step.”
Reducing the number of samples added to the reverse transcriptase qPCR (RT-qPCR) better conserves limited RNA samples. “For example, you may only have a small amount of patient blood, so the RNA is limited. Our kit requires only six to eight additional wells for each RNA preparation,” Dr. Kelley said.
While the kit doesn’t eliminate inhibitors, it provides valuable information up front. “Determining the presence of inhibitors helps the researcher identify early on if this is a problem. Then you can institute corrective measures to rescue the experiment (e.g., by diluting the RNA) or to re-extract using a better method. Once you have a reproducible method that has the least amount of carry-over inhibitors, you don’t necessarily have to assess for reaction inhibition every time.”