QRT-PCR/Single tube: Difference between revisions
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(Real-time RT-PCR moved to Quantitative reverse transcriptase PCR: naming here is difficult but I think RT RT PCR is confusing; real-time doesn't tell you much; Q tells you that this is all about quantification;) |
m (Real-time RT-PCR moved to QRT-PCR/Single tube: This is single tube qRT-PCR.) |
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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. | |||
== 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. | |||
== Protocol Endorsements == | |||
[[Category:Protocol]] | |||
[[Category:In vitro]] | |||
[[Category:RNA]] | |||
Latest revision as of 12:43, 9 October 2007
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.
