qPCR FAQs (Genomics Shared Resources)

• How can I learn more about the qPCR assays?
• Can I get a discount on reagents from ABI?
• How many replicate wells should I run per sample?
• Can I leave empty wells on my plate?
• Do I have a passive reference?
• How do I run a melting curve or dissociation stage at the end of an RQ run?
• Can I save my in-progress run data directly to Fred?
• Can I obtain Ct values from a Relative Quantification (RQ) run?
• How can I calculate ∆∆Ct from my Absolute Quantification (AQ) data?
• How often are background checks and calibrations run on the instruments?

Q: How can I learn more about the qPCR assays?

Applied Biosystems supplies on-line Tutorials for Real-Time PCR at the following website: http://www3.appliedbiosystems.com/AB_Home/Support/ TutorialsTroubleshooting/index.htm

Q: Can I get a discount on reagents from ABI?

You can receive a quote for discounts on Applied Biosystems products from either FHCRC buyer Peony Nhan-Cheng (206.667.6332;  pnhan@fhcrc.org) or Applied Biosystems Sales Representative Paula Sodora (800.248.0281 x7180; Paula.Sodora@appliedbiosystems.com).

Q: How many replicate wells should I run per sample?

It is recommended that all samples, including standard curve wells, should be run in triplicate.

Q: Can I leave empty wells on my plate?

You can leave empty wells on a sample plate, but the empty wells must be omitted under the set-up tab of your run document.

Q: Do I have a passive reference?

Many reagent kits are manufactured with a passive reference dye in the concentrated master mix. The instructions accompanying each kit will indicate if and what type of passive reference is added.

Q: How do I run a melting curve or dissociation stage at the end of a Relative Quantification (RQ) run?

The Relative Quantification (RQ) assay does not include a default dissociation stage setting.  If you require a dissociation stage after an RQ assay immediately upon completion of a run, create an Absolute Quantification (AQ) project and program the thermal cycler settings to run the default dissociation stage only for your sample plate.

Q: Can I save my in-progress run data directly to Fred?

You cannot save your in-progress run data directly to Fred.  The network connection is not suitable for saving in-progress data from the 7900HT equipment.  You must save your in-progress run to the D partition of the computer’s hard drive.

Q: Can I obtain Ct values from a Relative Quantification (RQ) run?

Ct values can be obtained from a RQ run by using the RQ Manager feature of qPCR SDSv2.3 software. For more information on how to use the software please contact Jenni Risler (206-667-4670; jrisler@fhcrc.org) or Elizabeth Jensen (206-667-4470; ejensen@fhcrc.org) for assistance.

Q: How can I calculate RQ from my Absolute Quantification (AQ) data?

The equation for RQ is:

∆Ct    = Ct (sample) - Ct (endogenous control)
∆∆Ct  = ∆Ct (sample) - ∆Ct (calibrator)
FC      = 2 ^-∆∆Ct  (normalized fold change relative to calibrator)

Q: How often are background checks and calibrations run on the instruments?

Background checks are run monthly on each qPCR machine. If elevated or problematic signal is noticed on a run, background checks are run more frequently. Block cleaning and spectral calibrations are conducted semi-annually. If heat block contamination is detected, block cleanings are conducted more frequently.

	FAQs