This facility was established by a grant from
Health Resources and Services Administration (HRSA)
|
|
|
|
|
Rapid Cycle Real-Time (kinetic) PCR using LightCycler instumentation (Roche)
Introduction / Quantitative RT-PCR / PCR Primer Design / Sample preparation / Amplification Data Analysis / References / Services and Fees / RT-PCR Services Request Form |
Expression levels of the housekeeping gene standards with known copy number are determined. For each unknown sample the levels of housekeeping gene and target gene are determined in separate reactions. Standard curves are generated for both parameters and are used for the quantification of the target and the housekeeping gene in each sample. For the target gene, the result is expressed as a relative ratio of the target divided by the housekeeping gene, thus normalizing each sample.
Data analysis for the concentration of target and reference gene is automatically achieved with the LightCycler software, but the calculation of the target/reference ratio has to be done manually or using a spreadsheet calculation program such as MS Excel. The fold change of “treated” vs “untreated” gene expression levels may then be determined by dividing the ratios of the target pair.
The Second Derivative Maximum Method and arithemetic baseline adustment is used. This method automatically calculates the fractional cycle number of the crossing point (a point on the amplification curve that always represents the same amount of PCR product in every curve) of each sample. This renders the method independent from user-born influences.