Quantitative PCR at UC Riverside Core Instrumentation Facility
UCR CIF provides access to two QPCR instruments, an Applied Biosystems (ABI) 7700 Sequence Detection System and a Bio-Rad iCycler iQ5. We provide individual and group training in general use of QPCR as well as specific use of our instrument, to include instruction in use of the plate setup and analysis windows of the software. We can help you sort through the amplicon detection and quantitation options available as well as other decisions that need to be made to get started doing experiments. UCR investigators pay a nominal charge for use of the instrument (Rates).
QPCR integrates PCR amplification of template cDNAs with fluorescence detection, recording accumulation of amplicons cycle-by-cycle. The 7700 and the iCycler iQ5 can be used for quantifying cDNAs investigators prepare from RNAs in their own labs ("two-step, two tube"), or they can be used for quantitative RT(reverse transcription)-PCR, in which the investigator combines the RT and PCR steps in one tube with no transfers ("two-step, one tube"). Please see our QuickStart Guides on the ABI 7700 and the Bio-Rad iCycler iQ5 for overviews of setup and data analysis on the two instruments.
Their are two basic options when choosing reporter fluorophore: The first, simplest and least expensive is the intercalating dye SYBR Green I, the fluorescence of which is very low in the absence of double-stranded DNA and very high in its presence. This dye has the virtue that it can be used for any PCR product. This virtue can become a pitfall if, under the thermal cycler conditions used, significant primer-dimer formation and extension occurs, since such products will also bind dye and contribute to signal. Both the 7700 and the iCycler iQ5 have the capability of automatically running an analysis of PCR product melting temperatures following the usual 40 PCR cycles for generating quantitative data; this allows the investigator to determine whether significant primer-dimer formation has occurred, since these products will have lower melting temperatures that are easily resolved from those of specific amplicons that are typically > 100 bp in length. Many people on campus use 200-reaction SYBR Green I kits that are available from a number of manufacturers including Applied Biosystems (cat. no. 4304886), Bio-Rad (cat. no. 170-8882), Qiagen (cat.no. 204143), Stratagene (cat. no. 600548) and Sigma (cat. no. S4438). These companies also sell kits for quantitative RT-PCR (two-step, one-tube procedures). Please be aware in using a commercial or home-made (see below) QPCR Master Mixs: if your master mix contains the passive reference dye ROX (usually in an amount sufficient to generate 1000-1500 a.f.u. per well), the 7700 software needs to be set to use ROX fluorescence to normalize reporter signal. If the mix does not contain ROX, you will need to turn off the ROX option in the software (under Show Analysis menu, select Instrument, Diagnostics, Advanced Options, Miscellaneous then unclick Reference). Not doing so can generate some artifactual fluctuations in software-calculated baseline fluorescence. The iCycler iQ5 does not use normalization of reporter signal. In addition to the commercial mixes, it is possible to prepare your own SYBR Green I PCR master mix from readily available reagents, an option that could afford substantial savings for bigger projects (QPCR home-made reagent mix). It may even be possible to use the PCR mix you routinely use in the lab, particularly if reactions are set up in a cold block to simulate the benefits of a hot-start Taq. This possibility is easily checked of course in the 7700 by running a dilution series and examining the amplification efficiencies (please see Tim Kingan for details). Other basic materials needed include PCR plates (e.g., Phenix MPX-PCR96-HS) and their optically transparent caps (e.g., AppliedBiosystems, cat. no. 4323032; these are strips containing 8 caps that seal one column in the plate).
The second and more sensitive option comprises one of a collection of fluorophore-labeled oligonucleotide probes (e.g., Taqman, Molecular Beacons or Scorpion probes), designed to anneal near the middle of an accumulating amplicon. Probes contain a reporter fluorophore, e.g., 6-FAM, typically at the 5' end, and a quenching fluorophore or "black-hole" (non-fluorescing) dye at the 3' end. Fluorescence in the absence of amplicon is low, because of intra-molecular quenching optimized in the design of oligonucleotide probes. Labeled probe is in excess of amplicon, at least during the exponential phase of amplification. During accumulation of amplicon, an increasing amount of probe hybridizes to target DNA. For the ABI Taqman probes, a "hydrolysis" probe, fluorescence then increases as the 5'-nuclease activity intrinsic to Taq polymerase hydrolyzes the 5' nucleotide (with fluorophore), freeing it from critical spacing with the quencher associated with intra-molecular energy transfer. Molecular Beacons are "hybridization" probes, in that their fluorescence increases on hybridization to accumulating amplicon, as quenching associated with their stem-loop structure is overcome.
See the Bustin reviews (below) and references therein for thorough treatments of these options as well as for considerations in the all-important decision on the use of absolute or relative methods in quantitiation of target DNAs.
UCR CIF makes available a Macintosh workstation with Primer Express, a single license primer and probe design application from ABI.
Following are web-based resources, available from ABI or posted on our server.
For AppliedBiosystems protocols on use of SYBR Green I in QPCR see:
http://docs.appliedbiosystems.com/search.taf?_UserReference=057A094D3B54E45F3F787F67
Download the following files:
4304965B SYBR® Green PCR
and RT PCR Reagents: Protocol: Rev B
4310251C SYBR® Green PCR
Master Mix and RT-PCR: Protocol: Rev C
Other useful downloads on QPCR, also on ABI's web site, can be found at:
http://www.appliedbiosystems.com/support/tutorials/
The software for analyzing data from the 7700, SDS 1.9.1, is freely available from Applied Biosystems and can be downloaded from:
http://www.appliedbiosystems.com/support/software/7700/updates.cfm
Your raw data files can be copied to Zip disc or jump drive and then analyzed on your lab Macintosh (no PC version available).
Available on our server are:
Excellent overviews of QPCR from S.A. Bustin:
| Bustin 2000 | Bustin 2002 |
A protocol for conducting a melting curve analysis following a QPCR run (lets you check for primer-dimer formation):
Dissociation Curve Analysis on the 7700
A PowerPoint presentation from a CIF workshop on QPCR can be downloaded at: