Real-time PCR

Contact: Jenni Risler, jrisler@fredhutch.org
Elizabeth Jensen, ejensen@fredhutch.org
Location: DE-341
Contact phone: (206) 667-4470
Contact fax: (206) 667-2825
Contact e-mail: ejensen@fredhutch.org

Lab Introduction and Consulting

The Genomics Resource provides access to self-service qPCR (real-time PCR) instruments located in DE-341. Four ABI QuantStudio5 Real Time PCR Systems (two 96-well block and two 384-well blocks) are available 24 hours a day, with staff available to answer questions Mon-Fri (9AM-5PM). See our FAQ on doing qPCR.

Those new to qPCR technology, ABI instrumentation, or the Genetic Analysis Lab should schedule a one-on-one introductory instrument training session. Please contact the resource staff to schedule an appointment.

All information provided below is pertinent to Fred Hutchinson/UW Cancer Consortium members. For non-consortium members interested in services please contact the Genomics Resource at (206) 667-4470 or one of the individuals listed below.

Contacts

  • For questions regarding services and related fees, please contact Jenni Risler at jrisler@fredhutch.org or (206) 667-4670.
     
  • For questions regarding services and related fees, please contact Elizabeth Jensen at ejensen@fredhutch.org or (206) 667-4470.

Reserving an Instrument

To reserve an instrument, those with a HutchNet ID and a fred account can use the online Instrument Scheduler. Those who do not have a HutchNet ID and fred account should contact the Genomics resource for assistance.

Evening hours must be scheduled by no later than 4PM the day of the desired reservation; weekend hours must be reserved by no later than 4 PM on the preceding Friday. If you must cancel a reservation, please give as much advanced notice as possible (no less than 24 hr). If you start your run late and it extends beyond the end of your reservation, please be aware that your run will be aborted to accommodate the next reservation in the queue.

Reagent and Consumable Purchases

All reagents and consumable are the responsibility of the investigator. Common suppliers include Life Technologies, Qiagen, Roche, Promega, Sigma Aldrich, Bio-rad, and Agilent. All consumables (plates, caps, film) must be of optical quality. A centrifuge with a microtiter plate rotor is available and is located in the vicinity of the instruments.

Results and Downstream Analysis

All data files will be saved on the hard drive of the computer operating the particular instrument. Please create a folder on the hard drive (C:/AppliedBiosystems/QuantStudio Design and Analysis/User File/Experiments) and designate that folder in the Save As query that appears when you begin your run. Please transfer the file to the server once your experimental run has been completed.

The ABI machines generate an .eds file which can be analyzed by QuantStudio Design and Analysis software for computers running Windows 7 or newer (PC-compatible only). Download a copy of the software . (License agreements dictate that some of the software is available only from FHCRC based computers. If you are not at Fred Hutch and need access to the software, please contact Genomics and we will arrange access for you.)

qPCR FAQs (Genomics Shared Resources)

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

Applied Biosystems offers an online education resource for Real-Time PCR at the following website: https://www.thermofisher.com/us/en/home/life-science/pcr/real-time-pcr/qpcr-education.html

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@fredhutch.org) or Life Technologies Sales Representative David Colgan ((800) 248-0281 x7444; David.Colgan@lifetech.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?

Yes

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?

A dissociation stage will be added to your cycling parameters by default if you select the Sybr Green option during plate set up

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. You must save your in-progress run to the C partition of the computer’s hard drive.

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.