Another use of qPCR is to assess the effects of gene expression or knockdown. “RT-qPCR is a valuable technique that can be used to ensure genes have been silenced following RNA interference studies,” Sarah Payne, Ph.D., product manager at TTP LabTech, said. This technique can be used to verify that a gene has been silenced following a positive hit obtained from a phenotypic assay run on Acumen® eX3.
“In one study, our instrument was used in a genome-wide siRNAi screen to characterize the implications of knockdown on a particular cell function and identify the genes involved in epidermal growth factor signaling. PCR techniques were used to confirm that the gene of interest had been silenced. This approach resulted in the successful identification of a number of genes involved in this signaling pathway. Ultimately, following up a positive hit identified by Acumen eX3 with RT-qPCR should result in a reliable, more time- and cost-effective method to distinguish false positives from true hits,” Dr. Payne said.
According to Joanne Franklin, Ph.D., marketing manager, the company is currently finalizing a new application for Mosquito®, its automated nanoliter liquid handler. “Mosquito is now being used to add samples and reagents to a PCR plate with a 1,536-well format that is utilized for miniaturized assays. This will result in reducing the costs of consumables and increasing sample throughput significantly.”
Scant amounts of sample can be a major limitation for deriving consistent and reliable data from qPCR. Increasing cost is another bottleneck. New tools derived from miniaturization and microfluidics are helping to solve these issues.
In olden times (~10 years ago), PCR technology analyzed up to 96 samples and required 20–50 uL reaction volumes. Fast-forward to the current time, a new instrument enhances throughput 16-fold more to 1,536 wells and needs a miniscule 0.5–2 µL per reaction. The LightCycler® 1536 Real-Time PCR system was recently released by Roche Applied Science.
According to Robertus Van Miltenburg, marketing director, “because of its miniaturization and efficiency, the new system is much more efficient in its use of sample and reagents. This is especially important because often only small amounts of precious sample material are available. Another important benefit is that the number of reactions per run can be increased, which both enhances throughput and decreases the need for plate-to-plate comparisons.”
Gudrun Tellman, Ph.D., product manager, described how the LightCycler 1536 System was recently utilized to perform expression profiling of human heart samples associated with cardiac disease. “We provided examples showing verification of microarray data. We obtained the same results when comparing the traditional lower throughput way or via the 1,536 format, illustrating the sensitivity and the reproducibility of our approach. Ultimately, this greatly enhances how much data you can get from your samples.”
The Roche system uses two excitation filters with two detection filters optimized for detecting mono- and dual-color hydrolysis probes as well as green intercalating dyes. The software facilitates easy set up and running reaction protocols related to the automation of high-throughput data analysis work flow.
New and exciting improvements in implementing qPCR are leading the way to advances in diagnostics, basic research, and the search for novel therapeutics. As sensitivity, scale, and scope broaden, most in the field are optimistic about the continued growth and expansion of qPCR technologies.