Quantitative+PCR

Quantitative PCR integrates PCR amplification of template cDNAs with flourescence detection, recording accumulation of amplicons cycle-by-cycle (1). A known qPCR template standard serves two main purposes. It functions as a positive control and as a reference for measuring the exact copy number of a transcript in an unknown sample(1). Quantitative PCR (QPCR, or real time PCR (rtPCR)) has emerged as a powerful virologic technique for measuring viral replication and viral loads in humans and animal models, in serum and in tissue samples, and both for culturable viruses and non-culturable viruses(2). Apart from increased sensitivity, this technique offers a high-throughput alternative to Southern blots for determining gene copy number, a significant factor when screening fungal mutants and transformants (3).

qPCR protocol: Preparation of the qPCR template standard 1. The qPCR template standard is provided as a dried pellet. Add 50 μ   l of d H2O to the tube and vortex briefly. The resuspended template is 10,000,000 copy/ μ   l. Thaw the 10 x dilution buffer tube from the kit at room temperature. 2. If the SYBR Green I method is used, reconstitute SybGREEN primer by adding 200 μ   l d H2O to  the primer tube and vortex it briefly. The resuspended primers are 10 pmol/ul each. (Omit this step if Taqman probe method is used) 3. Prepare 1.0 mL of 1 x dilution buffer by adding 100 μl of the 10 X buffer to 900 μl H2O. 4. Label six (6) Eppendorf tubes from 1 to 6. Transfer 90 μl of 1x buffer to each pre-labeled eppendorf tube (1 to 6). 5. Transfer 10 μl of template standard from the stock tube to eppendorf tube #1 (Fig. 2). Mix the solution by briefly vortexing. 6. Transfer 10 μl solution from tube #1 to tube #2, then mix the solution by vortexing.. 7. Perform remaining dilutions by repeating steps 5 and 6. 8. Transfer 5 μl of diluted template solution from each tube to a 96-well PCR plate (use 2 μl when a  384-well plate is used). Their are two basic options when choosing reporter fluorophore: The first, simplest and least expensive is the intercalating dye SYBR Green I, the fluorescence of which is very low in the absence of double-stranded DNA and very high in its presence. The second and more sensitive option comprises one of a collection of fluorophore-labeled oligonucleotide probes (e.g., Taqman, Molecular Beacons or Scorpion probes), designed to anneal near the middle of an accumulating amplicon. Probes contain a reporter fluorophore, e.g., 6-FAM, typically at the 5' end, and a quenching fluorophore or "black-hole" (non-fluorescing) dye at the 3' end (1).


 * Works Cited**

1. Bustin S.A. (2002). Quantification of mRNA using real-time reverse transcription PCR: trends and problems. Journal of Molecular Endocrinology 29, 23-39.

2. Crotty Shane, McCausland Megan M. (2008). Quantitative PCR technique for detecting lymphocytic choriomeningitis virus in vivo. J Virol Methods 147 (1), 167-176. 3. Solomon Peter, Simon V.S. Ipcho, James K. Hane, Kar-Chun Tan, Richard P. Oliver (2007). A quantitative PCR approach to determine gene copy number. Fungal genetic reports 55, 5-8.