The fast-paced, continually evolving field of quantitative PCR (qPCR) will be showcased at a CHI meeting in San Diego next month. Presenters will describe cutting-edge methodologies and emerging technologies such as digital microfluidics, nanopore and single-molecule sequencing, and improved ways to standardize data.
Traditionally, the first step in gene-expression studies is to purify RNA from samples. However, RNA isolation can be a problem for some applications. Gregory L. Shipley, Ph.D., assistant professor and director, Quantitative Genomics Core Laboratory, University of Texas Health Science Center, says that utilizing cell lysates for real-time qPCR is a helpful alternative. “Isolating RNA can be an expensive and time-consuming process. For small amounts of tissue or low numbers of cells, RNA purification isn’t practical.”
Dr. Shipley suggests that a better way is to utilize cell lysates directly for cDNA synthesis. “Lysates not only act as effective templates for amplification, they provide some added advantages. First, one can easily utilize a very small number of cells for real-time qPCR. If you tease out cell populations from a lung tissue sample, there are lots of cell types present. Typically, you might get a specific population of only about 20,000 to 100,000 cells using flow cytometry. It isn’t practical to isolate RNA from such a small number of cells, especially for analyzing multiple transcripts. Secondly, isolation of RNA can potentially skew the population whereas with cell lysates nothing is lost.”
According to Dr. Shipley, there are some drawbacks that must be taken into consideration. “When using cells directly, the benefit is that everything is in there. However, the disadvantage is that everything is in there. It is important to lyse cells in the presence of agents that will inhibit RNA degradation and to have a way to get rid of contaminating genomic DNA.
“Several manufacturers offer kits for making cell lysates for real-time qPCR and more are jumping on the bandwagon. The challenge for the future will be to figure out how to use small amounts of tissue directly for qPCR. Currently, this works less well in our hands.”