St. Curves w/ high Ct - Trying to assay low abundance mRNAs (Oct/09/2005 )
I just started working with PCR and am trying to measure the relative abundance of two target genes. We purchased predesigned Gene Expression Assays from Applied Biosystems for our targets, but the lowest Ct I've ever gotten from either of them is in the high 20's. I assume that just means my target is relatively rare, which is expected, but I am having trouble creating a standard curve for accurate measurements.
My 18S control curves look excellent, going from a Ct of ~15 to ~30, but these two targets are killing me. Should I try to design my own primer/probes or could it be something else? What're the steps to take to troubleshoot this?
I have already tried:
Isolating new/fresh RNA.
Increasing mRNA amount in One-step protocol.
Reducing serial dilutions for control curves from 1:10 to 1:5 or 1:4.
Switching to a two-step protocol.
But I've had absolutely no success, except for my really sexy 18s curves. Yeah, those look real nice. 
if you increase your template by a lot and you get very little decrease in Ct, you might want to look at your reaction efficiencies
i would get on the phone with ABI
i would get on the phone with ABI
Oh I am on the phone with them, but they seem to be trying to sweep me under the rug. Are there any other quality places to get predesigned, prevalidated primer/probes? I'm thinking of trying Roche's UPL if ABI keeps this up.
I feel your pain.
We had the best ABI rep in the world about 2 years ago when I first started doing real-time. he left about a year ago to work in another city; consequently I get to deal with their regular tech support people now if I am having an issue. needless to say I no longer recommend the company to new people. I think their products are usually pretty good but their support sucks. I suggested calling them because it really seems like you are up against a wall.
I do SYBR green; cheap and easy and no primer/probe combo. So, I cannot recommend anyone else. I do have one question; I know they're supposed to be all optimal and stuff, but do you still have to optimize your primer ratios and concentrations?
high Ct's might just be what you get. one of my genes runs between 26 and 33, depending on how I tweak the cells, and it is what it is....means you need to be a little more careful about reproducibility in your methods to keep your statistics tight, but it still works. the important factor is not getting the same Ct, but ensuring that your reaction efficiencies are tight. it's all relative.
good luck
They optimize it so you don't have to modify any ratios or concentrations. You can use their predesigned gene assays with their premade Universal Master Mix in your two step reactions and it should work fine.
My problem is, I can detect my targets with regular RT-PCR just fine. When I switch over to qPCR, using the relative abundance standard curve method, I get diddly squat.
Thanks for your replies though.
that doesn't make sense. about has to be a bad kit composition....sorry you are so frustrated!