Starting with semi quantitative RT-PCR and a little bit lost.. - (Mar/16/2008 )

Hi,

I'm starting some work on siRNA and semi-quantitative rt-pcr. All the primers and siRNA molecules I'm using are published on the literature..
For the rt-pcr I'm using Qiagen one-step RT-PCR kit and on my first attempt at siRNA transfection I didn't see any difference in the expression levels seen by the rt-pcr.

So what I decided to do was to try a new rt-pcr but using control cells (untreated) and decrease the amount of RNA template to see If i could see correlation between band intensity and starting RNA template. I used 2, 1.5, 1, 0.5 and 0.25 µg (total).

I ran the gel and I couldn't really see much difference. The 0.25µg yielded no product, but in the other 4 I obtained very similar bands. I did some rudimentary analysis using Photoshop and the histogram utility and the bands were within a 5% range of the 2µg reaction.

Maybe I'm using too many cycles in the PCR?
I used the same parameters that I read on the paper, the only difference is that they used a two-step approach.

Also, how do you quantify the bands? (is there a guide somewhere? i tried ImageJ but I can't seem to figure out how).

Thanks.

Here is a pic, from left to right
(1) 2 µg RNA
(2) 1.5
(3) 1
(4) 0.5
(5) 0.25

How many cycles are you running? I'm not sure cycle number is your problem though, since you get nothing with 0.25ug and your bands don't look saturated - if anything, you have less DNA in the 2ug lane.
Are you sure your RNA samples are clean and intact?

Some companies sell ladders with quantifying bands that would allow you to guess at amounts of DNA.

Hi
First, I think you should be doing a time-course, removing your tubes at, let's say, 20, 22, 25 cycles and so on, and proving that your PCR is in the linear range of amplification if you want to get a quantification. Also, I might be wrong about this, but I understood that conventional PCR has a dinamic range of 10, therefore it would probably be hard to see changes in the range you're interested in. Have you considered switching to real-time PCR?