Real Time PCR
Real-time PCR is a sensitive molecular biology technique for real-time monitoring of the amplification of target DNA sequence during PCR reaction. Real-time PCR can be used quantitatively and semi-quantitatively. Two common methods for the detection of PCR products in real-time PCR are non-specific fluorescent dyes that intercalate with any double-stranded DNA and sequence-specific DNA probes consisting of oligonucleotides that are labeled with a fluorescent reporter, which permits detection only after hybridization of the probe with its complementary sequence. During the annealing step of Real-Time PCR, a change in fluorescence is detected and it is interpreted as the cycle threshold value. A higher ct value means that the target molecule has a lower number of copies at the beginning of the PCR and a low ct value means a higher concentration of the target DNA molecule at the beginning of PCR. Real-Time PCR enables studying the expression level of target genes at the transcriptional level. Commercial kits are available that enables studying intermolecular interactions using conventional RT-PCR machine. It can also be used for augmentation and validation of the results obtained from high throughput technologies such as microarray and RNA-seq. DBT supported UDSCMAC facility at CIIDRET houses Applied Biosystems Step One Plus machine. This facility with state-of-the-art instrumentation serves national R&D and academic establishments having DSIR authorization and Customs exemption such as colleges, public labs, government foundations, and not-for-benefits associations. The facility has been exploited by several users across the country for their experiments.