Real-time quantitative PCR (RT-qPCR or qPCR) is revolutionizing many aspects of molecular biology and becoming the gold standard for accurate, sensitive, and fast quantification of gene expression. Relatively new, it also is experiencing growing pains.
Key challenges right now include finding the right reference gene and improving data analysis. As its popularity grows, new-kid-on-the-block technologies such as two-dimensional RT-qPCR and multiplex digital RT-qPCR are proliferating.
The ability to capture a two-dimensional (2D), spatially accurate expression profile of tissue provides a unique way to follow molecular pathologies. Michael Armani, Ph.D., developed, with his colleague Michael Tangrea, “a 2D-RT-qPCR methodology that quantifies RNA across tissue sections in a single platform.” Dr. Armani is a post-doctoral research assistant, lab of pathology, National Cancer Institute, and was a presenter at CHI’s recent “Quantitative Real Time PCR Conference for Molecular Diagnostics”.
“Overall, the purpose of this approach is the quantification of target mRNAs corresponding to their original positions within the two-dimensional tissue architecture,” he said. The researchers use a 384 well plate over which tissue sections are laid. Then through a sequential series of reactions, tissue is lysed, RNA isolated and reverse-transcribed, followed by qPCR.
“In collaboration with Elisabeth Smela and Benjamin Shapiro at the University of Maryland, we developed a method that uses a grid format in a multiwell plate to macro-dissect tissue sections in order to better preserve the spatial locations of the RNA. We developed and validated the 2D-RT-qPCR in 384-well plates using magnetic recovery beads for RNA isolation in a volume of 20 microliters. Use of such a small volume for tissue lysis and purification had not been done before. The first challenge for us was to characterize the purification efficiency.”
One of the major benefits and applications Dr. Armani envisions for this technology is the ability to mine RNA data in existing tissue banks to better understand pathologies and treatments. “Our end goal is to improve treatment decisions. Usually pathologists are microscopically studying and drawing conclusions on the type, grade, and prognosis for cancers. Although this is a 100-year-old technique, it is still the most actively used and robust method for diagnosis or treatment.
“Staining slides is inexpensive, consistent, and effective. However, considering that there are more than 20,000 genes that exist in humans, blocks of stored tissue essentially have locked up within them an incredible source of information particularly as to potential mutations and genetic patterns.”
The next goals for the project are to ramp up the number of gene-expression patterns. “Initially we provided proof-of-concept with one gene versus control. Then we did three, now we are up to 24 in each well. What’s needed next is to develop even finer resolution. ”