Hi, I have 3 genes (1st is my Gene of interest and two house keeping genes) each gene will have a forward primer , a reverse primer and a different Tm for each primer.
1-When doing the cDNA amplification step(since ill be using the Reverse and Forward primers for each of the three genes) based on what should I set the temperatures of the cycles ?
2- Do I have to do any changes in the qrt-PCR step (Since ill be using the same primers of question one)?
Thank you
Hi,
I do not really understand your experimental setup. If you want to quantify gene expression by quantitative reverse-transcription PCR I would recommend following:
1. cDNA synthesis random or poly.dT primed
2. qPCRs with gene-specific primers in single reactions using an aliquot of the cDNA
If you have a very limited amount of RNA you can do gene specific one-step qRT-PCR (combination of cDNA synthesis and PCR). However, you need probe-based assays for multiplexing or you have to run reactions for each gene separately.
Best,
Norman
hi Ayah Al Qaryoute
If I am understanding your question properly then multiple melt curve appears due to improper tm.
1. you will set or find the tm of each of the gene primers individually. Generally, Tm is based on the synthesis process. so when you get primers from the supplier, they will provide tm in the datasheet. Calculate the average of the particular gene FP+RP tm, divide them by 2 i.e finding avaerage. Then, take the average value, minus 5 deg. and plus 5 deg. from that average value, and FIRST YOU SHOULD GO FOR 2% agarose gel electrophoresis. Seeing the band intensity & gene expression pattern, you will need to figure its tm of the primers.
example: Gene A forward primer (FP): 58
Reverse primer (RP): 56
So, average: 58+56/2 = 57
you need to consider: 57, 52, 62. Mostly, its minus 5 which works. Now, upon running gel, you will see the band intensity and product size according to the ladder and decide the tm.
2. Each gene primers will have different tm so if you PCR machine has multiple tm setting to run the reaction with respective tm. otherwise, you will need to one tm in one run.
--Generally, housekeeping gene like GAPDH are high copy number, and their ct value range from 12-22 so you can run only GAPDH first with your sample and figure out the housekeeping gene expression pattern. for other genes, you will need to set tm and then go for qRT-PCR.
--Well, Primer preparation, working concentration, and Sample dilution also need to be looked to proper qRT-PCR amplification.
Regards,
Saurabh
Thank you Laurence for your answer.
Usually the setting I use to synthesis of cDNA from the RNA that I extracted is very similiar to what you suggested and in that step I never so far faced any problem specially that all using is the qscript cDNA super mix and RNA template (no primers are involed).
The problem am facing is in the qrt-PCR step when I have to use the forward and reverse primers for each gene cDNA in the 96-well plate and SyBER green (is am getting 3 prominant populations of Melting curves instead of one big curve.
Any suggestions based on your expertise?
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