How to Choose the Right PCR Reagents

Dr. Gregory Shipley, director of the Quantitative Genomics Core Laboratory at The University of Texas Health Science Center in Houston, discusses how he goes about evaluating and investing in new technologies and reagents.

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Dr. Gregory Shipley, director of the Quantitative Genomics Core Laboratory at The University of Texas Health Science Center in Houston, discusses with Tanuja Koppal, contributing editor to Lab Manager Magazine, the ways in which he goes about evaluating and investing in new technologies and reagents for carrying out real-time quantitative polymerase chain reactions (qPCR) for the various assays being conducted in his core facility. Dr. Shipley is an expert in designing qPCR assays and has written on the topic in various scientific journals and spoken on it at several conferences. He is also a very active member of various qPCR groups and listservs, where he has been known to offer his candid advice to novices and pros alike.

Q:How has the field evolved, and what are some of the new applications of the technology that you are working on?

A: Real-time PCR (RT-PCR) has been out there for nearly 14 years, and although people still want to do the standard transcript and gene quantification and analysis, there is a lot more you can now do with real-time qPCR instruments. One application driving the field is the direct use of cell lysates from a small sample or from a single cell to perform RT-PCR, without having to isolate the RNA. You can also do high-resolution melt (HRM) analysis, an application that’s going to grow more popular with time and is likely to take over the probebased single nucleotide polymorphism analysis for many assays. Real-time instruments can also be used for performing proximity ligation assays, which is a way to quantify proteins using qPCR for detection. This shows that an old dog can always learn new tricks. A realtime instrument is nothing but a high-end CCD camera coupled with a spectrophotometer and a thermocycler, and so there are a lot of ways you can leverage that to perform new kinds of assays. People are comfortable using probebased assays, since they have had a lot of experience with that, but it’s just a matter of learning how to make these new assays work.

Q:What would you advise for lab managers who have to continuously keep up with the new reagents and instrumentation being introduced into the market?

A:If you see a new reagent, I would suggest first talking to the salesperson and getting the person to send you a small aliquot to try. Then do some side-by-side comparisons with your old and new reagents, using the standards that you use for all your assays, and see if the new reagent works better or at least as well. If it has a better price, then it’s worth considering. The ready-made kits are a good deal because they save you time, but they cost more. So if you are operating small, then you may want to buy individual reagents and save money by making your own master mixes. However, if you are brand new to the game, then I highly recommend that you start with a kit, because you don’t want something going wrong because of the reagents.

Q:What about when evaluating new instrumentation or software products?

A:It’s the same thing. If you are serious about buying new equipment, you can get the company to bring in the machine for a week or so, to see how it works. If it doesn’t work for you or it doesn’t do what you need it to do, don’t buy it. Don’t buy a machine for what you need now, but buy it for what you think you might need in the future. If you buy an inexpensive machine or don’t buy the various software modules, then you may not be able to do certain things like running 384-well plates or multiplexing or HRM. Sometimes people buy new PCR machines with a certain project in mind, but what you really have to do is think ahead.

Q:What advice do you have for labs looking to negotiate a better price with various vendors?

A:Things have changed a lot in recent years. For instance, The University of Texas system has negotiated a certain price with vendors, and so every lab on our campus gets that same price. Other places are doing the same by leveraging their buying power. However, if you are in a small lab but can show that you are using a lot of reagents or if you can pool together with other labs to show purchasing power, then you can negotiate a better price. There are also “freezer programs” set up in many institutions where scientists can walk to and pick up reagents directly from the vendor-installed freezers or refrigerators, which saves money by eliminating shipping costs.

Q:What kinds of challenges do you run into when working with PCR reagents and how have you managed to overcome them?

A: In the late ’90s the Master Mix sometimes didn’t work and there were bad lots. But honestly, I can’t remember the last time in the recent past when there was a problem with the Master Mix. Part of what you are paying for with the ready-made reagents is the quality control that goes into the reagents before they come into the market. The same goes for other PCR reagents like primers, vectors and probes. One thing, however, that I would like to point out is that, although the companies that make oligonucleotides do their best to tell you how much reagent is in the tube, sometimes the amount varies. Hence, we always determine the exact amount of what is in the tube by looking at the optical density of the primers and probes. There is always some loss, especially after the reagent is dried down in the tube, and every now and then we get something that is almost tenfold less than what is expected. This is happening less and less than it used to, but, nonetheless, what’s on the label should be used only as a guide. There are also some problems with contamination. For instance, every primer and probe has some slight DNA contamination and you can pick it up with the assays that are really sensitive. (e.g., 18SrRNA) But there is nothing you can really do about it. Fortunately, it isn’t a problem for the vast majority of assays.

If you missed the Ask The Expert webinar “Utilizing Whole Cell Lysates Directly for Real-Time qPCR”, originally broadcast on Tuesday July 27, 2010, Click here to watch the archived video.

Categories: Ask the Expert

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Rethinking Green

Published: April 1, 2010

Cover Story

Rethinking Green

While the green movement is receiving less attention now than it has in recent years, it was able to take root with regulators who have become less tolerant of practices found to harm the environment. Many lab managers believe that adjusting their processes now may be more economically efficient and less disruptive to their work than racing to meet regulatory deadlines in the future.