PCR and Q-PCR

PCR Thermocyclers in the CGC

MJ Research Dyad Thermocyclers

The CGC has two MJ Research Dyad dual block PCR thermocyclers with interchangable PCR plate blocks, or Alpha blocks™. (Note that MJ Research was purchased by BioRad in 2005) There are both 96-well and 384-well Alpha blocks. 0.2 mL strip tubes can be used in the 9 Pair of MJ Research DYAD thermocyclers 6-well blocks, along with various PCR plates with conical tubes. These thermocyclers can be programmed to create thermal gradients of up to 20°C across the long axis (12 well axis) of the block.

Supplies/Costs

There is a $3.00 charge for using the thermocyclers. The CGC has Hardshell plates, generic flat top plates, and adhesive sealing film that can be purchased. Users can provide their own Taq mix and primers or cycle sequencing mixes, or they may purchase aliquots of these reagents from the CGC. See the CGC Store list for the details.

Practical Considerations

The CGC does not have a clean hood or workstation for setting up sensitive PCR reactions at this time. If you are doing contaminant sensitive reactions you should prepare your reaction plates or tubes under controlled conditions in your lab. It is also wise to assume the PCR machines in the CGC are "dirty" so you should clean the alpha block and lid to your satisfaction before begining 384 well DYAD a. block #1 g your reactions.

Remember to reserve time for your reactions on the online scheduling calendar! The two Dyad machines are referred to as "pcr_a" and "pcr_b" on the calendar, "a" being the one on the left. Each Dyad has two alpha blocks referred to as "block_1" and "block_2", on the left and right sides, respectively. All four of the alpha blocks which are normally installed accept 96 well plates. There are also two 384 well alpha blocks available for your high-throughput projects.

New to PCR? have a look at some of the external links here.
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Quantitative or Real-Time PCR in the CGC

The Chromo4 Real Time PCR Instrument

The CGC acquired an MJ Research Chromo4 Real Time 4-color 96-well PCR system in April 2005. This instrument is based on the MJ Research PTC 200 gradient thermocycler platform and it has all the standard features that other MJ Research thermocyclers have. The Chromo4 is operated by the Opticon Monitor 3 software on the connected Windows XP computer. The cycling programs are created in the same way as on the Dyad thermocyclers, except that it is done in a software w Opticon Monitor 3.0 software screen indow on the attached PC. The fluorescence data are collected by a scanning mechanism in the special heated lid. It can be used as a Real Time PCR machine, or as a standard PCR machine at times when all of the Dyad 96-well alpha blocks are busy.

Supplies/Costs

There is a $8.00 per run charge for using the Chromo4, to help cover the costs of maintenance and repairs. The Chromo4 uses the same 96-well PCR plates that the Dyads use. MJ Research recommends the use of white plates to maximize detection sensitivity, but the clear plates work well enough for most purposes and pipetting into them is easier. You also need either optically clear sealing film or optical flat snap caps to cover the wells and allow the fluorescence to be detected. Users can provide their own Taq mix, and/or SYBR green or purchase aliquouts from the CGC. Users are expected to supply their own primers. Software is available on the CGC computers that can be used to design primers. The CGC sells optically clear sealing film and PCR plates as noted above. See the Tips section below for further infomaration about plates.

Practical Considerations

Many facility users will use SYBR green for the majority of their quantitative PCR work. This is genereally the least expensive type of real time PCR reaction to run. Taq mixes with SYBR green already in the mix can be purchased from various suppliers, or you can use your favorite Taq mix and add the SYBR green from a stock solution yourself.
A typical SYBR green run with 40 cycles of PCR and a melting curve with data collected every 0.2°C will take about 3 hours.

The disadvantages of using SYBR green come from the fact that SYBR green will report the concentration of all double stranded products in the reaction, regardless of whether they are the desired product, primer dimers, or some other unintended side product. Probe based RT-PCR assays like TaqMan™ avoid this pitfall by using a specific probe sequence to report only the presence of your intended products. A melting curve is not required for most probe based assays, so these assays are faster than SYBR green assays as well. The only real drawback of the probe based assays is that one has to design and buy a specific labeled probe oligo for each gene of interest.

Applied Biosystems has a service and a software package for designing TaqMan™ probes and labeled primers, or you can consult the literature and design your own. The protocol archives page contains a listing of some of the more common fluorescent dyes which can be used for labelling primers or probes. BioRad has an online chart showing the spectrum of fluorescent dyes and the standard dyes that work in each optical channel of the Chromo4. When you access that page you have to choose "Chromo4" from the drop-down menu to see the Chromo4 optical channel information. When you are planning an experiment it is worth checking the quantitative PCR section of the protocols archive page to see if another user may have found a more efficient or less expensive way to do the kind of experiment you have in mind. The center posts useful protocols and user tips on that page when a user finds a method or experimental design they think is especially good.

Tips for Maximizing Data Quality

BioRad bought MJ Research in 2005, so BioRad is now the source of supplies for and information about MJ Research instruments. BioRad periodically conducts meetings on campus with Real Time PCR users where a local researcher presents a short talk on their work, people discuss the applicability of various quantitative PCR methods to their research, and share troubleshooting hints. The BioRad product specialists at these meetings also help users troubleshoot their problems and provide tips on improving data quality. The CGC has a copy of the book "Real-Time PCR, an Essential Guide", by Edwards, Logan,and Saunders, 2004, Horizon Bioscience.

From these meetings and from experiences of Q-PCR users in the CGC we have found that a properly fitting PCR plate and good plate/tube sealing are two things which are critical to success. BioRad recommends using low profile non-skirted polypropylene plates or low profile strip tubes as these seem to maintain the most uniform contact with the PCR block over the 30-40 cycles of an RT-PCR experiment. They also recommend using snap caps from the manufacturer of the plates to ensure reliable sealing. CGC users have also obtained reliable results using optically clear plate sealing film with a foam compression pad that has a small hole in the foam over each well. These foam pads come from Applied Biosystems, #4312639.
The clear low profile unskirted plate from MJ Research is catalog # MLL-9601. The white version is MLL-9651. Snap caps for these plates are TCS-0803.

Real Time PCR Background

A Very Brief Explanation of How RT-PCR Works

In a standard polymerase chain reaction the amount of product generated should, theoretically, double with each additional cycle. If that were really how it worked, then the amount of product present at the end would be a simple function of the amount of template present at the beginning. However, it doesn't work that way in real life. As the reaction progresses primers and nucleotides are consumed and eventually become limiting, so the efficiency per cycle starts to drop off at some point.

Real Time PCR can provide a quantitative relationship between the amount of template initially present and the amount of product formed because it provides a way of measuring the amount of product formed during each individual cycle using a fluorescent reporter dye of some sort. The basic principle is to measure the fluorescence signal at the end of each PCR extension step and plot the data. The plot will be sigmoidal, flat at the baseline level for the first 10-20 cycles while the amount of template initially accumulates RT PCR graph to the point where the fluorescence signal becomes detectable. Then the plot rises linearly for several cycles and finally begins to "roll over" and then becomes almost flat.

As you can see in the example graph at left, the detectable linear range is fairly narrow. Only in the linear range can you assume that there is a direct relationship between the amount of template present in the reaction and the intensity of the fluorescence signal. These measurements have to be made while the PCR is in progress, so this is called a "real time" measurement rather than an end point measurement. Thus the name Real Time PCR.

Quantitation

A standard curve can be made using dilutions of a known quantity of a reference template and this can be used to quantitate other samples. That is, assuming the amplification efficiency is the same for the test samples and the standards. The efficiency of each reaction can be calculated from the slope of the line during the linear phase of the reaction.

To make accurate comparisons between samples and standard curves, the PCR reactions for the samples and standards need to be optimized so that they come close to 100% efficiency. A melting curve can be made after the reaction is complete, as an additional check to ensure that only one product is being formed in the reaction. This is important to do in SYBR Green assays. The melting curve should have only one peak. Additional peaks or shoulders indicate that there is some non-specific priming or maybe primer dimer formation contributing to the fluorescence signal and throwing off the quanitation.

Gene Expression - relative quantitation

The basic idea is to compare the expression level of some gene of interest between cases where the organism or tissue has been subjected to different conditions or treatments, or maybe to compare the expression level in different tissues or parts of the same organism. To accomplish this you have to be able to normalize each test sample to the total amount of mRNA tested. A "housekeeping" gene, one that would be expected to be present in the same quantity in all the tissues tested, is usually used as a proxy for the total amount of mRNA in the sample. Of course, you can't do PCR with a single stranded template, so any gene expression experiment starts with production of cDNA using reverse transcriptase. There are one-step protocols and kits available to make the cDNA and then do the PCR in one reaction tube. As you might expect with a process like this that involves several steps where things can go wrong, having proper controls is very important. Even so, the sensitivity and precision of RT-PCR is far greater than that of the usual alternatives like Northen blots and in-situ hybridizations on thin sections. RT-PCR can also be done more quickly and doesn't require the use of toxic chemicals or radioactive probes.