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. ”
Multiplex Digital RT-PCR
Digital PCR provides a quantitative analysis of nucleic acids as it detects single molecules of DNA or RNA. “Compared to real-time qPCR, digital PCR provides absolute quantification of nucleic acids and potentially performs at a higher accuracy,” says Feng Shen, Ph.D., director of R&D, SlipChip.
“While digital PCR has been demonstrated on a variety of platforms, these platforms usually require complex control systems for sample manipulation and fluidic control. This could make digital PCR less routinely accessible in laboratories or resource-limited settings. The SlipChip is a microfluidic device that consists of two plates with imprinted wells, and the fluidic path can be established or broken apart by simply bringing wells on different plates in or out of contact. SlipChip can generate a large number of reaction compartments, which is required by digital PCR, by relative movement of the two plates, or a ‘slipping’ step.”
Targeting point-of-care and resource-limited settings, Dr. Shen and colleagues developed a multivolume digital RT-PCR SlipChip in Professor Rustem Ismagilov’s laboratory (currently at California Institute of Technology) to assess viral HIV and hepatitis C (HCV) loads. “Although the use of antiretroviral treatments has become more common, it is important to evaluate viral load from patients periodically to prevent the spread of drug resistance,” Dr. Shen explains. This requires quantitative measurements of viral RNA over a large dynamic range. We developed a microfluidic rotational SlipChip to perform multivolume digital RT-PCR with a dynamic range from 1.7×102 to 2.0×107 molecules/milliliter with threefold resolution and lower detection limit of 40 molecules/milliliter.
“Instead of using a large number of wells with uniform volume, we used far fewer wells with different volumes (from 0.2–625 nanoliters) to address the need of a large dynamic range. The tests were conducted using synthetic RNA and validated in a multiplex format by HIV viral RNA and HCV control viral RNA.”
The team found that the results of the viral load test on the SlipChip were self-consistent, and the HIV viral load tests of clinical patients’ samples were in good agreement with the results determined by the Roche COBAS Ampliprep/COBAS TaqMan HIV-1 Test.