- 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
- Technique chosen:
RT-PCR: Real time PCR or Quantitative PCR (qPCR)
- What does this technique ‘do’?
Amplification and detection of DNA simultaneously in Real Time.
- 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
- 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 (peer-reviewed 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.
- 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.
- 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.
- 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: 151-159.
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.
Our revised question:
Which of the following statements are TRUE?
A) One advantage of real time PCR is that it can be performed at a single temperature without the need for specialized equipment.
B) If a DNA polymerase other than Taq polymerase was placed in the TaqMan qPCR reaction, the target sequence will be amplified but it will not be detected by the machine.
C) In an experiment, we want to find a mutation in our gene of interest by melt curve analysis, we can use SYBR Green method of qPCR.
D) Use of gel, low sensitivity and poor precision are all draw backs of traditional PCR
E) Contamination by DNA fragments with no sequence homology to the target sequence can show up as a false positive in TaqMan method but not in SYBR Green Method.
Link to powerpoint presentation: https://docs.google.com/presentation/d/150EOQskwstkJAW_c45nkrYdH_an7zSkDElTfQb3a3Dg/edit#slide=id.gbd6576bcc_0_25
Reflection:
I chose our techniques project as one of my top assignments as I felt like it was different from all the others since it was the first assignment that really forced us to work on something as a collective group. Starting with our research for the qRT-PCR, I learned to search and extract relevant information literature effectively and most importantly simplify all the technical details of the method so that it could be better understood. As a group we were able to identify key characteristics and traits of the technique to include in our presentation and we tried to communicate that information clearly during the actual presentation by outlining key facts and points we found to be of particular importance. This project also let me assess my own knowledge of qRT-PCR which was introduced to me in BIOL 335 and I found that I had to relearn the procedures for the technique to fill in any gaps from what I could remember. Overall, this project was a great group effort and really taught us how to collaborate and work together as a group for the rest of the semester. We also established a facebook group as a way to communicate our ideas and collectively work on assignments if we couldn’t meet in person. I also really enjoyed the Techniques Café part of the project in that it allowed us to see what other groups had done and how they chose to present their projects.