Quantitative Real-Time PCR

qPCR

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Quantitative Real-Time PCR

 

QuantStudio 12KFlex with 384-well, 96-well and available OpenArray block is in C2705.

Please schedule your run if using 96-well plate project, as our usual block is the 384-well plate block.

384-well barcoded plates and seals may be purchased in the Supply Center.

You may call  x5823 or (preferred) email Rob  or Georgina for instructions.

You may still use SDS software to create the plate and  export the setup file into the "Incoming 7900 Plates" folder in the network folder located here:

\\hersheymed\files\Research\CoreFacility\Results\Functional Genomics Incoming

Note:  File - Export setup, not the SDS file itself.

Analysis for Comparative Ct plates (RQ) can be done in your lab using free ExpressionSuite software from LifeTechnologies.

 

 

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.

SYBR Green

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.

Flanking 5' (forward) and 3' (reverse) Primers can be made in our Macromolecular Core Facility -- or through external companies such as IDTDNA, Invitrogen, Applied Biosystems, etc.