Real Time PCR method comments - (Jul/29/2009 )
I am new to Real Time PCR, however i am using it for a number of experiments. If i describe my method below i wonder if anyone would be so kind as to give me some feedback.
The aim of the RT-PCR is to determine the relative levels of a particular mRNA in E. coli between a treated sample and a non-treated control sample.
1. I isolate RNA and treat with DNase, checking for DNA removal by performing a Taq PCR and seeing no product.
2. I quantify the RNA approximately using a spectrophotometer (nanodrop)
3. I add approximately equal amounts of RNA from the treated and non-treated samples to reverse transcriptase PCR reactions. These are primed using random 6-mers.
4. I set up Real Time PCR reactions using SYBR green master mix. I set up reactions using cDNA from my treated and non treated samples using 2 primer sets. One set is for my gene of interest, the other is for an internal reference gene for which expression does not change under the conditions of the experiment.
5. I run the RT-PCR and obtain values for Ct and reaction efficiency for each reaction.
6. DATA ANALYSIS (the bit im not sure about)...I ensure that the reaction efficiencys for the PCR reactions set up with treated and untreated sample cDNA are roughly the same (+/- 5% max difference but usually only around 1-2% change). I then take the Ct values and calculate delta delta Ct using:
(Ct(gene of interest)-Ct(internal reference gene))
I then include the factor of amplification efficiency (Ae) and calculate Ae^delta delta Ct and provide the value as n in 2^n
If anyone could comment on this i would be very grateful. Critisism would be welcome as i am keen to learn from this
Thankyou in advance,
That is the correct assumption for a delta delta Ct experiment. Congratulations on a well-prepared protocol flow and good luck to you.
You might want to add a step of running your RNA on a denaturing formaldehyde gel before you do reverse transcription(RT) . Check for RNA integrity first.
Other than that the general flow is alright. Remember to prepare NTC (no tempplate controls) for your real-time.
Don't forget to do a meltingcurve when you use sybr green.