Quantitative Real Time PCR (Low- to Mid-Throughput)

Real-time Quantitative PCR is the reliable detection and measurement of products generated during each cycle of the PCR process that are directly proportional to the amount of template prior to the start of the PCR process. To accomplish this it is necessary to have a method of detecting the accumulation of PCR product and an instrument in which to perform the thermocycling that is adapted to record the results during each PCR cycle. This can be achieved with a number of instruments that are commercially available. The real-time QPCR instruments currently available to Core users are two ABI 7900 HTs.

Sybr Green, TaqMan or hydrolysis probes, or hybridization probes measure the fluorescent change per PCR cycle. The fluorescent change is plotted versus time, represented by cycle number, to produce a continuous measure of PCR amplification. To provide precise quantification of initial target in each PCR reaction, the amplification plot is examined at a point during the log phase of product accumulation. This is accomplished by assigning a fluorescence threshold above background and determining the point at which each amplification plot crosses the threshold (defined as the threshold cycle number or Ct). Differences in threshold cycle number are used to quantify the relative amount of PCR target contained within each tube. Assuming that each reaction functions at 100% PCR efficiency, a difference of one Ct represents a two-fold difference in the amount of starting template.

Taqman Relative Expression

Description
TaqMan qPCR allows users to compare the expression of genes of interest with selected control genes.  We generally run all reactions in triplicate on 384-well plates, so numerous samples and assays can be run simultaneously.  Standard control genes and a variety of assays are available through the Core; these are listed in our appendix. Hundreds of assays are readily available through other companies, or the Core can help you design a unique assay. Results are provided in an Excel document, detailing CT, dCT, relative expression values, and running conditions.

Science
Taqman chemistry (generically, a hydrolysis probe) relies on the 5’-3’ exonuclease activity of Taq polymerase, which degrades a hybridized non-extendible DNA probe during the extension step of the PCR. The basis for this system is to continuously measure PCR product accumulation using a dual-labeled fluorogenic oligonucleotide probe, called a TaqMan probe. This probe is composed of a short (ca. 20-25 bases) oligonucleotide that is labeled with two different fluorescent dyes. On the 5’ terminus is a reporter dye and on the 3’ terminus is a quenching dye. The oligonucleotide sequence is homologous to a target sequence in the genome and PCR primers encompassing the probe sequence are added.

When the probe is intact, energy transfer between the two fluorophors occurs and the quencher quenches emission from the reporter. During the extension phase of PCR, the probe is cleaved by 5’ nuclease activity of Taq polymerase releasing the reporter from the oligonucleotide-quencher and resulting in an increase of reporter emission intensity. Computer software examines the fluorescence intensity of reporter and quencher over the course of the amplification to calculate the increase in normalized reporter emission intensity continuously.

Minor Groove Binders (see Minor Groove Binders (3’ moiety, used in ABI Taqman assays) (see reference below) or Locked Nucleic Acids (used in the Roche Universal Probe Library) (see here and here) can decrease the size of the probe by increasing the annealing temperature. Smaller probes are more specific due to the decreased probability of mis-priming, and therefore are more desirable.

Protocol
Available here.

Pricing
See pricing schedule here.

What to Provide
Please provide the core either with cDNA of known concentration or with RNA of known or unknown concentration. (If submitting RNA, please read Reverse Transcription / What to Provide.)

The total amount of template needed for a specific project can be calculated by determining the number of reactions needed and multiplying by 5ng/rxn in the following way:

[(# of genes)(triplicate) + 3] * 5ng/rxn = minimum quantity

Example:
[(5 genes)(3) + 3] * 5 = 90ng of template needed

This is the MINIMUM amount of template needed.  If you have more volume to spare, please provide it!  Also, for projects testing more than 5 genes or with an increased input per reaction, excess template is required.  If you provide the core with insufficient template to complete your project a processing charge will be applied.  Please call Core Staff with any questions.  Remember to submit the appropriate order form on CoreAdmin.

Reference
Kutyavin I.V., et al (2005) 3’-Minor groove binder-DNA probes increase sequence specificity at PCR extension temperatures Nucl. Acids Res. 28: 655-661


Sybr Green Relative Expression

Description
Sybr Green chemistry is a popular alternative to Taqman chemistry and in some cases may be a more economical route. The Genome Core processes cDNA or RNA samples provided by the user and will generate relative expression data for a number of user-specified test genes, which are then compared to a single or many control genes. Projects are run similar to Taqman relative expression protocols except that they utilize the Sybr Green dye and generally include a DNAse treatment before cDNA synthesis. Users are charged per reaction on a 384-well plate.

Science
SYBR Green I dye intercalates into double-stranded DNA and produces a fluorescent signal. The intensity of the signal is proportional to the amount of dsDNA present in the reaction. Therefore, at each step of the PCR reaction, the signal intensity increases as the amount of product increases. This provides a simple and reliable method that may be more sensitive and less expensive than Taqman chemistry.  However, it lacks the specificity of Taqman chemistry leading to problems with non-specific amplification caused by primer dimers and DNA contamination.

Protocol
Available here.

Pricing
See pricing schedule here.

What to Provide
Please see Taqman Relative Expression / What to Provide (above). For Sybr projects we will use extra volume of the most abundant sample to run a No RT Control.  Be sure to determine whether you would like to run a DNAse treatment.  Remember to upload the appropriate order form on CoreAdmin.

MicroRNA Relative Expression

Description
The Genome Core uses Applied Biosystems Taqman miRNA Assays to determine the expression of specific miRNA transcripts relative to a control miRNA gene.  Users provide samples containing total RNA along with the Taqman miRNA Assay requested for testing.  The Core will generate relative expression data for the requested miRNAs of interest and send the results to the user in a simple Excel format.  Users are charged per sample (cDNA synthesis) and per reaction on a 384-well plate (relative expression).

Science
Micro RNAs are a class of non-encoding small RNAs (21-23 base pairs in length) that play a role in gene regulation. MiRNAs use the RNA interference pathway to influence gene regulation by cleaving or repressing the translation of their messenger RNA targets.  Because of their small size, miRNA specific primers are needed in the reverse transcription step to increase the size of the miRNA template before the quantitative PCR step.  As a result, each miRNA must be separately reverse transcribed using the Core’s protocol. 

Protocol
Available here.

Pricing
See pricing schedule here.

What to Provide
Please submit the RNA samples you would like tested in STRIP-TUBES labeled with a Sharpie.  You may not submit cDNA for MicroRNA relative expression.  RNA must be at a concentration of 1 ng/ul.  Make sure to provide enough sample to complete your project and include excess volume to account for pipetting steps.  Remember to upload your order form on CoreAdmin.

gDNA Copy Number Analysis

Description
The Genome Core can use Taqman Chemistry methods to determine the genomic copy number of a particular gene or test locus in a sample.  Users provide the Core with genomic DNA samples (often including a known copy number control) and indicate which gene they would like to test for copy number variants.  Results are provided in a spreadsheet containing all experimental data and graphs detailing the determined copy number of the test locus in each of the samples.  Users are charged per reaction on a 384-well plate.

Science
gDNA copy number is measured by comparing the genomic region of interest to a control locus that is present in a stable copy number and is measured similar to relative expression analysis.  The Core Staff can recommend control genes specific to your study organism and has some available.  Users with new projects are encouraged to test more than one control locus for their study. The best results are achieved with high quality DNA and known copy number control samples. Taqman chemistry is often a preferred choice for this kind of study due to the increased specificity provided by the probe.

Protocol

Pricing
See pricing schedule here.

What to Provide
Please provide the core with your gDNA samples in 1.5ml eppendorf tubes labeled numerically with Sharpie. ALL SAMPLES SUBMITTED FOR COPY NUMBER ANALYSIS SHOULD BE AT THE SAME CONCENTRATION +/- 1NG/UL.  The total amount of template per sample needed for a specific copy number project can be calculated by determining the number of reactions needed and multiplying by 10ng/rxn in the following way:

[(# of genes)(triplicate) + 3] * 10ng/rxn = minimum quantity

Example:
[(3 genes)(3) + 3] * 10 = 120ng of template needed

This is the MIMIMUM amount of template needed and you will need to provide extra for the dilution steps required for copy number analysis.  If you have more volume to spare, please provide it!  If you provide the core with insufficient template to complete your project, a processing charge will be applied.  Please call Core Staff with any questions.  Remember to submit the appropriate order form on CoreAdmin.

DNA Quantification

The Genome Core can quantify genomic DNA using real-time PCR, a standard curve, a CA (SSR) repeat probe, and a pool of primers. Users specify which of the Genome Core primer pools, such as ovarian, prostate, or breast pools and provide freshly isolated and diluted genomic DNA samples, a control sample at a concentration of 1ng/ul, and a standard, usually at 3.1ng/ul. From the provided standard, the Genome Core performs a serial dilution to create the standard curve. From the known standard curve the amount of unknown genomic DNA can be determined. The sensitivity of this quantification method makes it ideal for FFPE (Formalin Fixed Paraffin Embedded) tissue. Since the ideal running conditions require freshly isolated genomic DNA, the Genome Core requires 2-day advance notice to schedule DNA quantification experiments. Data is provided in a spreadsheet format and users are charged per reaction well on a 384 well plate.

Science
This method uses quantitative microsatellite analysis (QuMA) for rapid measurement of relative DNA sequence copy number. In QuMA, the copy number of a test locus relative to a pooled reference is assessed using quantitative, real-time PCR amplification of loci carrying simple sequence repeats. For studies on tumors, 5-6 control loci are chosen specifically to avoid chomosomal regions known to be affected by the cancer of interest. Pooling of multiple loci helps avoid bias by any single locus.  These loci pools can be used as controls for gDNA copy number studies as well. For more information, please consult the following reference:

Ginzinger, D.G. et al (2000). Measurement of DNA Copy Number at Microsatellite Loci Using Quantitative PCR Analysis. Cancer Research 60, 5405-5409.

Protocol
Available here.

Pricing
See pricing schedule here.

What to Provide
Please provide the core with the DNA samples to be tested in STRIP-TUBES labeled numerically with a Sharpie.  You must submit a MINIMUM of 40ul of each sample.  Make sure that one of your samples is a control sample containing 12 ul of DNA at 1ng/ul.  You are also required to provide the core with an aliquot of DNA Standard at 3.0ng/ul OR 3.1ng/ul of which a minimum of 40ul is also required.  Please label the tube and your order form with the concentration provided.  Remember to upload the appropriate order form on CoreAdmin and to specify which control genes/test genes you would like run.

Additional Resources
A searchable database of core facilities at all UCSF campus locations, provided by the Clinical and Translational Science Institute at UCSF, is available here.