QRT-PCR/Single tube

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.

Starting Materials

• 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)

Basic Principle

• 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.

Protocol

• 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
  • END
• 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.

Analysis

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.