No success generating standard curves...

[donchoi]

Hello, I am trying to do some real-time RT-PCR. I am currently using the One-Step SYBR Green Kit from Qiagen, and detecting using an MJ Opticon DNA Engine.

• I have done the one step reaction in a standard (non real-time) cycler, and I see very specific products using my primer sets. I have checked annealing Temp optimization, and am using Ta=58C (though it works higher as well).
• I ordered standard pooled reference human total RNA from Clontech, and am using that as the template for the standard curves.
• I have set up mRNA standards from 100 ng to 1 pg in 10x serial dilutions, and also generated some additional dilutions in-between to get more data.
• When I run the real-time standard curves, I get inconsistent Ct values which can vary by 3+ Cts (even among the duplicates of the same reaction).
• My NTCs are very consistently non-amplifying.
• It appears that at 100 ng I am saturating the RT reaction, and I don't see additional product at that concentration.

This is my first shot at solo real-time RT-PCR. Any advice would be appreciated!

[Aga]

So you have the right primers.

Originally Posted by donchoi

When I run the real-time standard curves, I get inconsistent Ct values which can vary by 3+ Cts (even among the duplicates of the same reaction).

Have you tried to eliminate pippetting errors (you probably have)? The problems may occur during serial dilutions of your standard (it may happen with small volumes).

Originally Posted by donchoi

It appears that at 100 ng I am saturating the RT reaction, and I don't see additional product at that concentration.

It happened to me once, but even with lower concentration (50ng). You won't get any additional product at this concentration.