About Quantitative Real Time PCR
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.
Taqman or Sybr Green measure the fluorescent change per PCR cycle. The fluorescent change is plotted vs 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 relative cycle number, or Ct). Difference in the Ct are used to quantify the relative amount of PCR target contained within each tube. Assuming that each reaction functions at 100% efficiency, a difference of one Ct represents a two-fold difference in the amount of starting template.
Sample requirements for Taqman and Sybr Green:
Please provide either RNA (if wishing to use the DNase and RT Core Services) or cDNA of a known concentration. If providing cDNA please provide a no RT control, if providing RNA we will use extra volume of the most abundant sample for a no RT control.
The amount of cDNA necessary can be calculated by determining the number of reactions and number of GOI (assays) and multiplying by 5ng/rxn. All samples are run in triplicate.
(No. of assays x triplicate) + 3 x 5ng/ul = minimum cDNA quantity
This is the minimum amount of template needed, if you have more volume to spare please provide it. If you provide the core with insufficient template to complete the experiments requested a processing fee will be charged.
Results of qPCR are provided in an excel document, detailing Ct, delta Ct, relative expression values and running conditions. If Dnase and RT treatments were also performed these details will also be included.
DNA quantification is a method of quantifying genomic DNA using real-time PCR to generate a standard curve from a serial dilution. From the known standard curve the amount of unknown genomic DNA can be determined. This quantitation method is more sensitive and specific to PCR-able DNA than either Nanodrop or QUBIT quantitation.
The sensitivity of this method makes it ideal quantitation of DNA samples with low quality and concentration, such as FFPE (Formalin Fixed Paraffin Embedded) tissue or CTCs (circulating tumor cell). This quantitation method is also used for determining the concentration of pre-library prep samples for sequencing.
MicroRNAs are small (~22 nucleotides) endogenous RNAs, that play important regulatory roles in animals and plants by targeting mRNA transcripts for cleavage or translational repression. Because of their small size, they require special primers for reverse transcription. This increases the size of the miRNA in order to fit primers and a Taqman probe required for gene expression analysis.
In the reverse transcription (RT) step, cDNA is reverse transcribed from total RNA samples using specific miRNA primers. The miRNA RT differs from regular RT in that it requires primers specific to the miRNA of interest. Each sample must have an RT reaction using the specific miRNA RT primer. This requires ABI’s MicroRNA Reverse Transcription Kit, a set of RT primers, and a Taqman (TM) expression assay from ABI.
Please only submit RNA samples, we cannot accept cDNA for miRNA relative expression due to the requirement for miRNA specific primers in the RT step. RNA must be at a concentration of 1ng/ul, please include additional volume to allow for pipetting steps.
If primers are custom designed by the user, it is essential to test the efficiency of the binding of these primers before attempting to determine relative gene expression levels. We use a set of serial dilutions and create a standard curve to compare the efficiency of the binding of the designed primer set to one of our control sets at varying concentrations.
We require one positive control and one no RT sample to test for PCR efficiency. Please provide RNA or cDNA at 100ng/ul or greater in a minimum of 15ul volume per primer set.
Sybr Green chemistry is a popular alternative to Taqman chemistry. Projects are run in a very similar way to Taqman relative gene expression, except that Sybr Green dye is utilized.
Sybr Green I dye intercalates into double-stranded DNA (dsDNA) 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 volume 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.
Taqman qPCR enables the comparison of expression levels of genes of interest (GOI) to the expression levels of one or more endogenous control genes. The expression level is measured as a “Cycles at Threshold” (CT)value -- the cycle in which the florescence level crosses a threshold value of fluorescence, which occurs during the exponential phase of amplification. This CT value is directly correlated to the amount of starting transcript and therefore can be used to calculate relative amounts of starting transcript.
TaqMan chemistry uses two PCR primers (a forward and a reverse) and a probe, which anneals to the middle of the amplicon. On the 5’- end of the probe is a florescent reporter molecule, typically FAM. On the 3’- end is a quencher molecule, typically Black Hole Quencher 1 (BHQ1) or TAMRA. While both the reporter and quencher are attached to the probe, the quencher inhibits reporter fluorescence. During PCR extension, however, the reporter is cleaved from the probe and released from the quencher. This allows the real-time PCR instrument to detect fluorescence from the reporter molecule. BHQ1 quencher probes normally have a dark purple color, while TAMRA quencher probes are bright pink. For the analysis, it is important to know which quencher was used.
Using Taqman chemistry methods the genomic copy number of a gene or test locus in a sample can be determined. Taqman chemistry is the preferred choice for this type of study due to the increased specificity provided by the probe. 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 has a set of control genes and recommend that new users test more than one control gene for their study. The best results are achieved with high quality DNA and known copy number control samples.
Please provide all samples for testing at the same concentration +/- 1ng/ul. The total amount of template per sample can be calculated below:
[(# of genes x 3 (triplicate) + 3] x 10ng/ul = minimum gDNA quantity
This is the minimum amount required, if you have spare sample please provide it. If you provide insufficient template to the core to complete your project a processing charge will be applied.