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This protocol describes one-step real time quantitative reverse transcription PCR to quantify relative levels of a particular mRNA sequence between two samples. This technique is also called known commerically as Taqman, qRT-PCR, real-time PCR. This particular protocol describes use of a flourescent labeled probe (FAM) to provide readings.
- Validated PCR primers & probe that are efficient over the range of RNA that you are assaying. Primers are typically designed with a 58 degree Tm but may vary by lab.
- PCR primers and probe for a house keeping gene (ie B-actin, GAPDH, RPL19)
- Taq Polymerase, MuLV RT
- dNTPs, MgCl2, nuclease free water
- PCR strip caps or 96-well plates with transpearant caps
- total RNA (~50ng per reaction, diluted to 10ng/ul)
- Combine RT, Taq, dNTPs, primers, probe and RNA in a single tube. Add enzymes last.
- Reverse transcribe the RNA for ~ 30' to form a template for Taq-mediated PCR.
- Do 40 cycles of PCR. Measure the levels of template after each cycle with a real-time PCR machine.
- Analyze the results.
- Generate a 10x solution for each gene target that includes the appropriate forward primer, reverse primer, and labelled probe (FAM-BHQ1, FAM-TAM, etc).
- Combine RT, Taq, dNTPs, MgCl2 and H20 in a single tube called your master mix. Make sure to add enzymes last. Mix by pipetting (do not vortex enzymes).
- Add the appropriate quantity (refer to your kit) of master mix to 10x primer mix.
- Dilute RNA to ~ 10ng/ul. Pipet 5 ul into each well.
- Add PCR reaction mix for each gene target into the appropriate well.
- Cycles for MuLV reverse transcriptase with Amplitaq Gold from Applied Biosystems. This program may need adjustments depending on your primer design. It's a good starting point.
- 30' at 48 degrees
- 10' at 94 degrees
- 40 Cycles
- 30" 94 degrees
- 60" 60 degrees
- (optional) 60" 72 degrees
- The first time you use a primer set it is a good idea to run the sample out to ensure that you're getting one band, thus showing that your primers are specific for your desired gene product.
Delta-Delta Ct Method. See Livak KJ, Schmittgen. Methods 25 402-408 (2001)
Comments & Tips
- Use a master mix to ensure a consistent amount of enzyme in each tube that you will be comparing.