Rhode Island Genomics and Sequencing Center

Rhode Island Genomics and Sequencing Center

College of the Environment and Life Sciences

220 Morrill Science Bldg.

University of Rhode Island

Kingston, RI 02881

Helpful Tips for Successful QPCR and QRT-PCR using SYBR Green

Generating reliable data by QPCR and QRT-PCR requires careful preparation of the assay and may require optimization of primer concentrations to achieve efficiencies close to 100%. Following are some tips that have been employed in the Nelson Lab for developing successful QPCR and QRT-PCR assays.

I.  Primer Design:

·  Primers should be designed to amplify about a 100 bp sequence. The Nelson Lab typically uses the SciTools on the Integrated DNA Technologies (IDT) website (www.idtdna.com) for primer design.

·  Once primers are obtained they should be checked by traditional PCR and electrophoresis to confirm that only one product is produced. This PCR product can then be used for generating a standard curve, or it can be cloned into a vector of choice if a standard curve using a plasmid is preferred.

·  If more than one band is generated with a primer pair then it may be necessary to alter the annealing temperature or design a new primer set.

II.  Template Preparation and Quantification:

·  It is important to use a “clean” template for standards and unknowns. PCR products for standard curves should be purified prior to use. The Nelson Lab uses column-based kits from Qiagen (Valencia, CA) for PCR purification, and DNA and RNA extractions.

·  There are several methods that can be used to quantify your nucleic acid. Two common methods are UV spectrophotometry and fluorescence (Picogreen for DNA or Ribogreen for RNA). The Nelson Lab typically uses the Nanodrop UV spectrophotometer which only requires 2 ml of sample. Whatever method is used (UV or fluorescence) it is important to be consistent and not switch back and forth between quantification methods if data from multiple experiments is going to be compared.

·  For QRT-PCR it is important to determine the integrity of your RNA sample prior to doing any expression analysis. The Nelson lab evaluates RNA integrity by separating 2 mg of RNA on a 1% ethydium bromide agarose gel, and evaluating the rRNA bands. Non-degraded bacterial RNA will produce 2 sharp rRNA bands (16S and 23S), whereas RNA contaminated with RNases will be produce smeared rRNA bands. There is also a more sensitive technique for evaluating RNA integrity using the Agilent Bioanalyzer which is available in the RI-INBRE Core Facility, but requires the purchase of specialized cartridges for the instrument.

III. Standard Curve Preparation:

·  Standard curves can be generated using purified PCR products or plasmids containing the sequence of interest. Typically, the Nelson Lab generates standard curves from purified PCR products using 107à101 copies/rxn, but standard curves with a smaller range can be prepared if you know approximately how many copies to expect in your unknowns.

·  Standard curves can be generated in nuclease-free water or nuclease-free water containing 100 ng/ml yeast tRNA (Sigma-Aldrich; St. Louis, MO). The yeast tRNA acts as a carrier molecule and adheres to nucleic acid binding sites in the microcentrifuge tube, preventing binding of the nucleic acid of interest. This is especially important for the lower points on the standard curve where binding of your nucleic acid to the tube can affect the kinetics of the reaction. In addition, we have found that the yeast tRNA allows for multiple freeze-thaw cycles of the standard.

·  The Nelson Lab generates standard curves in nuclease-free water containing 100 ng/ml yeast tRNA. Typically, 100 ml of each standard is prepared and aliquoted in PCR strip tubes to facilitate transfer to the QPCR plate with a multi-channel pipette. The remaining material is stored at -20°C for future use. Therefore, 10 standard curves can be run using this preparation. This increases reproducibility from experiment to experiment as a new dilution series does not have to be prepared each time an experiment is run.

·  When preparing the standard curve it is critical that care is used when opening and closing tubes to prevent contamination of your gloves or the work area with aspirate from the tube. PCR reactions contain on the order of 1011 copies/ml; therefore contamination of the work area is a big concern. If possible standard curves should be set up in a work space separate from where unknown and no template reactions are set up.

·  Once a standard curve has been generated it should be tested prior to running any unknown samples. The standard curve (run in duplicate) should generate an R2 value close to 1.0 and efficiency close to 100% (+/- 10%). The Nelson Lab typically uses 50 nM of forward and reverse primer the first time an assay is run.

·  If the efficiency falls outside the acceptable range (90-110%) then primer optimization or a new primer pair may be needed. The first thing to try is to increase or decrease your primer concentration by 2 or 4 fold and see if the efficiency improves. If the efficiency is below 90% increase the primer concentration. If the efficiency is above 110% decrease the primer concentration. Further primer optimization may be required where the concentrations of the forward and reverse primer are different. For more information on this technique see the “Quantitative PCR A-Z” book available in the GSC.

·  Sometimes no amount of primer optimization will result in an acceptable efficiency. In this case it is necessary to start over and design new primers.

IV. Sample Preparation of Unknowns:

·  The concentration of unknown samples (DNA or RNA) needs to be determined by the user as it is based on the number of copies of the sequence of interest. The Nelson Lab typically uses 10 ng of nucleic acid per reaction (5ml of 2ng/ml unknown sample), but this may need to be adjusted after initial experiments.

·  Once an unknown concentration is determined to fall on the standard curve, all samples are diluted to this concentration so that results can be compared.

·  It is important to dilute unknown samples in the same diluent as that used to prepare the standard curve. We have found that using nuclease-free water containing 100 ng/ml yeast tRNA allows us to freeze-thaw diluted samples multiple times without loss of signal.

·  For QRT-PCR using bacterial RNA it is critical that the sample be free of DNA contamination. The Nelson Lab treats all bacterial RNA samples with DNase (Promega; Madison, WI) followed by a column clean-up (RNeasy kit; Qiagen) to remove the DNase prior to QRT-PCR.

·  For QRT-PCR using eukaryotic RNA primers can be designed to span introns so that minimal DNA contamination will not affect the assay, and DNase treatment is not needed.

V.  For more information and tips see:

·  http://www.gene-quantification.info/

·  qPCRlistserver – Yahoo! Groups

Created by Ryan Rhodes: June, 2008