I would really appreciate your support as this is my first introduction to the real time PCR. I am performing relative gene expression using power up syber green and on a biorad cfx instrument.
Imade my run twice and each time I am getting no amplification curves at all ( not even a single line) and no CT record, and very weird melting curves. I am attaching the photo.
I extracted RNA from treated and untreated cells and my RNA conc was around 1500ng/ul and purity of 1.6.
I reverse transcibed 600ng in a total 20ul reaction volume so my cDNA conc was 30ng/ul
I made a 10ul total volume of my real time PCR reaction; I tried different amounts of the cDNA starting from 10ng until 90 ng
I tried two different genes and one control.
The primers amount for all of them were 500nM.
I don't know why I am not getting any CT data! Not even early or late and not even good or bad curves where I can optimize from! I am getting a blank amplification curve and a weird melting curves!!
Could anyone advise what possible problems I have ? Is it something related to the machine? Or the set up? Or the master mix compatibility with the device?
And what does it mean that I am getting melting but not amplification curves? I understand that the melting curves are the opposite of the amplification curves and whatever got amplified first (even a primer dimer) should be dissociated at the end and gives something! So how come I am getting dissociation but not amplification curves!
Alaa Youssef Farag I think there is problem in the primers because the melting curve peaks are not sharp and unique that means there is non specific amplification . what is the size of your amplicon for the control gene and the target gene?!
@abrar Jamous
Around 220 pb. But again how come I am getting no amplification curves at all?
"The primers amount for all of them were 500nM."
Why did you choose thing number for primer conc? Some people may disagree, but for my experiences that number is not necessary. Is that the stock concentration? I used to work with 100 nM primers. Increasing primer conc could increase primer dimer issues. Also, you can achieve primer dimer and amplification at the same time (you would see two seperate peaks at different Tms), i don't recall seeing primer dimers exlusively most of the time.
I also couldn't discern the units on your amp curve on the attached picture and i'm not familiar with its scale (i used CFX96 before). Are you running 35-40 cycles? CFX96 should work with SYBR green channel. Do you have a positive control sample that you've amplified previously? It's always good to have a reference sample in case you need to troubleshoot (just make a cDNA that is equally pooled from all your other cDNAs). If there are other people in the lab using this machine, ask them if they're getting results which should rule out machine-related issues. Lastly, what is your cDNA dilution from the original cDNA synthesis?
Dear Can,
The protocol for the master mix I am using (power up syber green) gives a range of 300-800 nM for primer conc. , So I choose 500 nM as a first trial.
If it is as u say, I am not getting neither primer dimer nor significant amplification! I am getting blank amplification curve!! The thing that I couldn't entrpre! No CTs, no curves, no background signal, nothing!!!
I am running 40 cycles according to the protocol. And unfortunately I don't have a positive control as this is my first time doing the real time PCR thing!
The machine should be working! However, I didn't set up the plate myself. Do you think that a problem may happened in the set up of the plate??
I didn't dilute my cDNA ! I used exactly the amounts that I have mentioned!
Alaa Youssef Farag I hope you have validated the primers once by simply setting up a normal PCR using cDNA as template and checking it on gel. If you are getting a good band on gel then I would conclude that primer concentration is not a problem. Since you did not diluted your cDNA at all I should expect some amplification even if expression of your gene of interest is not high.
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