Introduction to Real-Time PCR
Techniques for monitoring global patterns of gene expressions have rapidly advanced. Microarrays allow for comparison of thousands of mRNAs among different RNA samples. However the method does not provide absolute quantitative analysis of gene expression. Real-time PCR, on the other hand, does.
Real-time PCR allows for more precise measurement of a limited number of genes in a large number of samples. Once candidate genes have been identified by microarray screening, the responses are confirmed by the use of Real-time PCR. While microarray screening is useful for testing the responses of large numbers of genes, Real-time PCR gives more precise measurements and is much better adapted to analyses of large numbers of samples.
Real-time PCR provides rapid and precise confirmation of gene changes. Though there are several different approaches to Real-time PCR, we recommend the use of SYBR Green detection (Molecular Probes). There are several reasons for this. For one, multiple reactions can be set up rapidly and inexpensively with standard oligonucleotides. Also, Real-time PCR relies on the fluorescence quantification of PCR product during each amplification cycle. Other detection systems such as molecular beacons and TaqMan assays rely on the synthesis of a fluorescently labeled detection oligonucleotide. Although these have the advantage of specificity they are more costly and time-consuming--the latter caused by the delay in obtaining the fluorescently labeled oligonucleotide.
When added to the reaction mix, SYBR Green binds to the hybridized dsDNA and fluoresces. During denaturation, it dissociates from the strand and fluorescence decreases. During the extension phase SYBR Green begins to bind again to the dsDNA. At the completion of polymerization, the SYBR Green is completely bound and fluoresces accordingly. SYBR Green allows the reaction to be based on standard oligonucleotides. Because SYBR Green will detect any PCR product, including nonspecific products and primer-dimers, care must be taken during oligonucleotide design.
Our primers are 20-mers with a 55% G-C content and a single 3' -G or -C. They are tested for specificity by BLAST (http://www.ncbi.nlm.nih.gov/BLAST/) and for folding and self-annealing by standard DNA analysis software. Primer pairs are tested for specificity and absence of primer-dimer formation by PCR followed by gel electrophoresis.
Real-time PCR requires the use of a specialized thermocycler with fluorescence detection capability. We use the ABI PRISM 7900HT which can perform assays in the 384-well format.
Although commercial kits are available, we use our own design, assembling our own reaction components.
For more information on real-time PCR and its applications, see:
- Monitoring G-protein-coupled receptor signaling with DNA microarrays and real-time polymerase chain reaction, Methods Enzymol. 2002. Vol.345:556-69
- Accuracy and calibration of commercial oligonucleotide and custom cDNA microarrays, Nucleic Acids Res. 2002. Vol.30:e48.
The ABI PRISM 7900HT
The ABI PRISM 7900HT Sequence Detection System (SDS) is designed for high throughput detection of fluorescent PCR-related chemistries. The instrument is currently configured to use 384-well plates and is capable of unattended 24 hour operation using a robotic arm to process plates in the queue.
It is capable of real-time, end-point, and dissociation curve analyses. A Microsoft Windows-compatible computer coordinates the Sequence Detection instrument, the bar-code reader and the automation module. The software saves the raw data from individual samples, whole plates or batches of plates for analysis. More information on the instrument can be found at Applied Biosystems' Web site at: http://home.appliedbiosystems.com. Because this machine is extremely delicate and complex, only qualified members of the Quantitative Genomics Facility will be able to operate it. You will however, be able to analyze your own data. A copy of the software for data analysis will be supplied to you for your convenience.
SYBR Green Protocol
Reaction Components (10µl per reaction)
|Deionized water||2.3 µl||230 µl|
|PCR buffer 10X: 200 mM Tris-HCl
(pH 8.4), 500 mM KCl
|MgCl2, 50 mM||1µl||100 µl|
|SYBR Green 1, 100X*||0.05 µl||5 µl|
|dNTPs, 10 mM each||0.2 µl||20 µl|
|Primer mix, 5 µM each||0.4 µl||40 µl|
|Taq DNA polymerase, 5 U/µl **||0.05 µl||5 µl|
|Sample||5 µl||500 µl|
* The 100X SYBR Green 1 is prepared by diluting 100 µl of the stock 10,000x concentrate into 10 ml of DMSO, then stored in 0.5 ml aliquots at -20º C.
** "Hot start" Taq should be used. We use Invitrogen's Platinum Taq, but you can experiment with other brands until you find the one that works best for you.
Once the mix is ready, add 5µl into each well of the 384-well plate that will contain a sample.
Follow by adding 5µl sample to each of the same wells and mix by repeat pipetting. To bring contents to the bottom of the wells, spin covered plates briefly (1 - 2 min.) in a benchtop centrifuge fitted with a microplate holder. Plates are then placed in the thermal cycler and run as follows:
- 95º C for 2 min. to activate enzyme †
- 95º C for 15 sec. to denature
- 55º C for 20 sec. to anneal
- 72º C for 30 sec. to extend
- Repeat last three steps for a total of 40 cycles
† Time will vary according to enzyme used.