TECHNIQUES SPEED-DATING: Quantitative Polymerase Chain Reaction

Names and contributions of group members:

Kenrick Ocampo-Tan (Editing, answering questions)

Natasha Tripp (added a few bullet points, edited references)

Lorenzo Luis Casal (editing, answering questions)

Melissa Chen (Editing, condensing information, poster design)

Kimmy Wong (Organized documents, added several bullet points, editing info)

Technique chosen: Quantitative PCR and its derivatives

What does this technique ‘do’?

• Quantitative polymerase chain reaction estimates the starting copy number of a DNA/mRNA segment
  • Can be estimated using the final product concentration after ‘X’ cycles (end-point qPCR) or by detecting the amount of products generated after each cycle (real-time qPCR)¹

What applications are this technique employed for?

• Estimating DNA copy number or detecting DNA from a sample²
• Gene expression analysis by quantifying mRNA (RT-qPCR)^2,4

What questions (give a couple of examples) relating to gene regulation and/or development can be addressed using this technique?

• How does the copy number of a certain gene compare with others? (qPCR)
• What housekeeping genes are active during specific time in development of fetal animals? (RT-qPCR)
• What genes are used in development but rarely expressed in adulthood? (RT-qPCR)
• Is there differential expression of genes under different stress conditions? (RT-qPCR)

What critical reagents are required to use this technique?

• Both qPCR and RT-qPCR require standard PCR reagents (Forward and reverse primers, dNTPs, thermophilic polymerase, Mg2+, appropriate buffers)
  • qPCR also requires DNA template and either a dye or probe that fluoresces when hydrolyzed or hybridized⁴
  • RT-qPCR requires reverse-transcriptase (and other standard RT-PCR reagents) in addition to all of the above
• real-time PCR requires a PCR machine capable of detecting the amount of product after each cycle

What critical information is required to be able to employ this technique?

• Sequence data to be able to create primers
• Proper annealing temperature for reaction specificity

References:

1. Heid, Christian A., Junco Stevens, Kenneth J. Livak, and P. Mickey Williams. “Real time quantitative PCR.” Genome research10 (1996): 986-994. Web. 01 Feb 2015.
2. Karlen, Yann, Alan McNair, Sébastien Perseguers, Christian Mazza, and Nicolas Mermod. “Statistical significance of quantitative PCR.” BMC bioinformatics1 (2007): 131. Web. 01 Feb 2015.
3. Lederman, Lynne. “QPCR.” BioTechniques4 (2009): 817-19. Web. 01 Feb 2015.
4. VanGuilder, Heather, Kent Vrana, and Willard Freeman. “Twenty-five Years Of Quantitative PCR For Gene Expression Analysis.” Biotechniques 5 (2008): 619-26. Web. 01 Feb 2015.