Download Real time PCR or Quantitative PCR

BIOL 463
A Technique in Five Minutes
TECHNIQUES PRESENTATIONS: WRITE-UP TEMPLATE
1. Names and contributions of group members:
Alanna: Research, Write-up, Powerpoint
Susan: Research, Write-up, Powerpoint
Rachel: Research, Write-up, Powerpoint
Jane: Research, Write-up, Powerpoint
Sam: Research, Write-up, Powerpoint
2. Technique chosen:
RT-PCR: Real time PCR or Quantitative PCR (qPCR)
3. What does this technique ‘do’?
Amplification and detection of DNA simultaneously in Real Time.
4. What applications is this technique employed for?
● Gene expression analysis or mRNA analysis
○ Hein et al. (2001) employed real time PCR to analyze murine
gamma interferon-gamma mRNA (a cytokine mRNA) expression
in splenocytes
● Detection of GMOs in food
○ Zeitler et al. (2002) identified real time PCR as a method for
quantifying transgenic contaminants with herbicide resistance in
conventional rape seed.
● Cancer or disease detection
○ Multiplex real-time reverse transcriptase PCR is an applicable
method for the detection, identification, and quantification HBV,
HCV and HIV-1
○ Bernard and Wittwer (2002) used real-time PCR for detection of
multiple breast cancer molecular markers
● Genetic variation analysis
BIOL 463
A Technique in Five Minutes
○ Real-time PCR is able to detect mutations in the sequence,
including single nucleotide polymorphisms (SNPs) through its
melting point analysis.
5. What questions relating to gene regulation and/or development can
be addressed using this technique? Provide two examples (peerreviewed papers) that use this technique.
Cytokine gene expression and regulation during infection:
Example: Th1 and Th2 cytokine gene expression in primary infection and
vaccination against Fasciola gigantica in buffaloes by real-time PCR.
Cytochrome expression and regulation in different life stages (unfertilized
eggs, embryos/larvae and adult tissue) and its role on neurodevelopment and
plasticity:
Example: Real-time PCR analysis of cytochrome P450 aromatase
expression in zebrafish: Gene specific tissue distribution, sex differences,
developmental programming, and estrogen regulation.
6. What critical reagents are required to use this technique?
● Taqman based real-time PCR
○ Primers, Taq polymerase, Mg2+, Nuclease free H2O, dNTPs,
Reverse transcriptase (if starting material is RNA), Taqman
probes, appropriate buffers and DNA template.
● SYBR Green based real-time PCR
○ Primers, thermophilic DNA polymerase, Mg2+, SYBR green I dye,
Nuclease free H2O, dNTPs, Reverse transcriptase (if starting
material is RNA), appropriate buffers and DNA template.
● Other materials include: Real-time PCR machine that is able to complete
thermal cycling steps for PCR and collect fluorescence data
simultaneously, a computer, and suitable computer software for data
collection and analysis.
BIOL 463
A Technique in Five Minutes
7. What critical information is required to be able to employ this
technique?
● Sequence data on the target sequence to design proper probes and
primers.
● Melting temperatures for PCR products for melting curve analysis to
determine amplification specificity.
8. References:
Bernard PS, Wittwer CT. Real-time PCR technology for cancer diagnostics. Clin
Chem 2002; 48:1178–1185.
Bustin SA, Mueller R. Real-time reverse transcription PCR (qRT-PCR) and its
potential use in clinical diagnosis. Clinical Science 2005; 109:365-379.
Hein J, Schellenberg U, Bein G, Hackstein H. Quantification of murine IFN-γ
mRNA and protein expression: impact of real-time kinetic RT-PCR using
SYBR Green I dye. Scand J Immunol 2001; 54:285–291
Kumar N, Raina OK, Nagar G, Prakash V, Jacob SS. Th1 and Th2 cytokine gene
expression in primary infection and vaccination against Fasciola gigantica
in buffaloes by real-time PCR. Parasitol Res 2013; 112:3561-3568.
Sawyer SJ, Gerstner KA, Callard GV. Real-time PCR analysis of cytochrome
P450 aromatase expression in zebrafish: Gene specific tissue distribution,
sex differences, developmental programming, and estrogen regulation.
General and comparative Endocrinology 2006; 147:108-117.
Valasek MA, Repa, JJ. The power of real-time PCR. Adv Physiol 2005; 29: 151159.
Zeitler R, Pietsch K, Waiblinger H. Validation of real-time PCR methods for the
quantification of transgenic contaminations in rape seed. Eur Food Res
Technol 2002; 214:346-351.