Potts Lab qPCR for dummies (and undergrads who want to learn more about what's going on in those experiments they are doing!)

Lets learn a little about qPCR!

Tradition PCR - DNA is amplified, and following amplification DNA is visialized by electrophoresis, which allows us to see qualitative differences in DNA based on fragment size.

Realtime PCR - DNA is amplified using instument that allows the monitoring of the amount of DNA created during each amplification by use a a fluorogenic system. Being able to track the amplification through the exponential phase allows us to use real time assays to measure the quantity of target DNA in the sample by following the rate DNA is produced during amplification.

  • First here's a refresher on traditional PCR... PCR
  • And some qPCR basics... qPCR vs traditional PCR

    • The assays our lab does use the SYBR Green qPCR method, as it is relatively inexpensive and doesn't require the synthesis and optimixation of specialty probe sets.

  • A cute SYBR green animation... SYBR Green Animation
  • More detail on SYBR based PCR... SYBR Green qPCR

    • Designing a good qPCR assay is not trivial, there are many factors such as template quatlity, assay type, controls and standards used, that effect accurate quantification.

  • More extensive reference on qPCR... qPCR Guide

    • For Friend virus, we can use genomic DNA to measure proviral (viral DNA integrated into the mouse genome) titers, or RNA to measure actual viral RNA in the cells.

    ***more links to come below***

    Of course PCR doesn't work on RNA, so before doing qPCR the the RNA must be converted to cDNA. This can be done in a step separate from the qPCR using reverse transcriptase (RT) methods that turn all RNA into DNA so multiple PCR assays can be performed on the same sample, or with specific primers that make DNA from the region of interest.

    Reverse transcriptase synthesizes DNA strand complementary to single strand RNA beginning at a double stranded staring point. Three methods for creating that double stranded point are used.

    1. Oligo dT primers can bind the polyadenylated tail of mRNA
    2. Random hexamer can bind and create random priming spots along the RNA strand.
    3. Specific primers can bind to the region of interest.

    Alternatively, if specific primers for the region of interest are used, the RT step may be part of a 2 step qPCR where a RT step to create a cDNA strand from the RNA precedes the actual amplification steps of the qPCR assay. cDNA &rtPCR