Quantitative Real-Time PCR
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Quantitative Real-Time PCR
For researchers at the Hershey Center for Applied Research we are trying to make things easy for you.
- The SDS2.2.2 software is on a computer in the Freeman lab, room 3200.
- 384-well plates and covers may be be available in room 3200. They may also be purchased through the Supply Center.
Sign onto the computer with your e-pass -or the person currently signed on lets you create your file.
You will set up the plate as if you are running it on the instrument, and type the barcode into the initial window that you would normally scan into.
Save the SDS file you create into the "Incoming 7900 Plates" folder in the network folder located here:
\\hersheymed\files\Research\CoreFacility\Results\Functional Genomics Incoming
For 384 well plates : Save as -- YourName_PI_BARCODE.
The plate is then put in the refrigerator in room 3200 and we collect them once a day around 1:30 PM.
NOTE: if you miss the 1:30 deadline, the plate can wait until the next day and/or you can bring it over to College of Medicine, room C2705 by 4:30.
---- Create the file and save it on the Functional Genomics Incoming folder on the network drive. We pull that file up and run the plate and put the file - now with data - back onto the network in the Functional Genomics Results folder in the individual researchers' folder the next day for you to analyze (again, on the computer in Freeman lab / 3200).
Quantitative Real-Time PCR is used to very precisely measure the amount of gene expression in cells or tissue of interest, relative to an endogenous control gene such as 18s, b-actin, etc. The two major methods to perform QRTPCR that are supported here in the Functional Genomics Core Facility are dual-labeled probe chemistry (TAQMAN®) and SYBR green chemistry.
Our recommendation for ease of use is to utilize the pre-optimized Primer/Probe Applied Biosystems Gene Expression Assays ( aka Assays-on-Demand ). These may be ordered through the Core Facility.
We can assist in finding other sources should your gene not be available at ABI, either with another company or with design assistance ( see below on this page ).
A very good source of information describing techniques and including troubleshooting may be found in the 2006 GenomeTech-RT-PCR TechGuide
A brief description ( of both Taqman 5'-nuclease ( dual-labelled probe chemistry ) and SYBR green ) follows:
Taqman Dual-Labeled Probe ( 5'-nuclease )
An oligo that is complementary to a portion of your gene of interest sequence between your PCR primers. This oligo has an fluor attached to the 5' end, and a quencher attached to the 3' end. When the oligo has hybridized to your gene sequence, and the polymerase incorporates it into the new product, the fluor is allowed to distance itself from the quencher. The rate at which new copies of your gene of interest are generated is inferred by the rate at which the intensity of this free fluor increases.
Dual labeled probes are more specific than SYBR green because dual labeled probe chemistry requires specific amplification of the gene of interest in order for fluorescence to be generated. SYBR Green, however, will generate fluorescent signal in the cases of mispriming and the formation of primer dimers. In addition, two different genes of interest may be amplified in one reaction, and detected independently from one another by using different fluors one each dual labeled probe. The disadvantage of using dual-labeled probes is that one dual-labeled probe must be purchase for each gene of interest.
A dye that will bind to double stranded nucleic acid. As your primers anneal and the polymerase extends to make new copies of your gene of interest, the amount of SYBR green increases proportionately. The rate at which new copies of your gene of interest are generated is inferred by the rate at which SYBR green fluorescence increases.
Should you be using an unavailable species and/or your gene is not yet listed we are also available to assist with Primer Design and Purchase.
A frequently updated list of validated primer/probe sets for quantitation of a variety of mRNAs is maintained at http://www.realtimeprimers.org/
To design your own primer/probe sets, you can use Primer Express, which is available in our facility (C2705).
Contact Rob Brucklacher (x5823) for more information and assistance.