Quantitative Real Time PCR

Please complete and attach the qPCR full service form and upload to MyCores when placing the service order

 

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

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.