Gourmet chefs know that getting the right combination of ingredients is what makes or breaks a recipe. So do life scientists. Nowhere is this lesson more apparent than in real-time PCR. Requiring a relatively precise balance of components, this quantitative technique can often stymie researchers’ efforts to assess gene expression. The introduction of master mixes spelled relief for many; however, while improving reproducibility and efficiency, master mixes also come with their own set of stipulations. A good dose of experience and wariness can allow you to take full advantage of the advertised benefits.
“Kits are designed for one-size-fits-all. Clearly, that’s not the case in the life sciences,” said Kevin Knudtson, PhD, director of the DNA Facility at the University of Iowa in Iowa City, Iowa. With master mixes, “there are times when you have to do some optimization or when using a custom mix is warranted.”
The explosion of the market for—and selection of—master mixes stemmed from the tedious preparation process of PCR. Into each tube, researchers had to add a precise amount of several ingredients. Contamination loomed close with the transfer of liquids from source to reaction tube. The bigger threat was perhaps human error. Slightly more or less pressure applied to the dispenser could mean critical changes in volume. Or, with a moment’s lapse of attention, a researcher could entirely overlook a tube or two.
Containing most of the necessary reagents, master mixes vastly reduce the chance for the experiment to go awry. The blend includes water, buffer, deoxynucleotides (dNTPs), and polymerase. Formulas made for conventional PCR can include dyes for use with gel electrophoresis, which usually follows amplification. Solutions geared toward real-time PCR, or quantitative PCR (qPCR), contain fluorescent dyes that allow for detection during cycling. Master mixes come in various concentrations, along with nuclease-free water for diluting to the strength that you need. You can also find lines that offer a gradient of buffer strengths and those that are designed specifically for hot start protocols. The preparation is complete when you add your sample templates and the appropriate primers.
While you’ll find that most products are pre-optimized as promised, the basic laws of biology will sometimes require your own tweaking. For example, template targets rich in adenine and thymine may require you to alter the dNTP ratio, Knudtson said. GC-rich targets are notorious for forming hairpin structures. Adding a relaxing agent, such as dimethyl sulfoxide, prevents such occurrences. You may also find yourself running a few reactions with different buffer concentrations in order to determine which allows the polymerase to function at its best. In fact, studies have shown that “the DNA polymerase-buffer system used for quantitative analysis can impact the performance of the system, and when used to quantify unknown samples it affects the accuracy of the data,” according to a paper published in the January 2004 issue of the Journal of Clinical Microbiology. (P Wolffs et al., “Impact of DNA Polymerases and Their Buffer Systems on Quantitative Real-Time PCR,” Journal of Clinical Microbiology, 42(1):408-411, January 2004.)
Starting out by experimenting with the individual components before purchasing a master mix could be a good strategy, suggests Eric Rappaport, PhD, director of the Nucleic Acid/Protein Research Core Facility at the Children’s Hospital of Philadelphia. “Once I’m comfortable with the template concentration, then I switch over to the master mix for the convenience,” he said. Otherwise, with commercial assays, or for those with published protocols, the pre-mixed solutions work well the majority of the time.
They work so well that Knudtson asserted that he “can count on one hand the number of times that a homemade mix worked better than the commercial master mixes.” While clearly a non-scientific estimation, Knudtson said he has more confidence with the pre-made solutions than the skill of most researchers who use the core facility. And, after ensuring the viability of the reverse transcriptase reaction, which transcribes RNA into DNA, “everything else seems to fall in line,” he said.
Give it a try with products such as those below.
