Real-time PCR questions (Mx3000P) - Need some help with interpretation (Jul/23/2005 )
Hello all,
I'm an undergraduate studying a novel soybean gene. I'm doing transcript localization studies and I've performed real-time PCR using SYBR Green just as a qualitative way to monitor changes in transcript levels thoughout plant development.
My first question regards configuring the Plate Setup in the Mx3000P software. For my positive control I set the well type as "Unknown". After the run was over, however, I realized that I should have set this to "Standard". This shouldn't make a difference in the level of fluoresence though, right? I'm asking this because my amplification plot shows my positive control peak as hardly above the Ct. I've used this positive control before though with positive results. Perhaps it was just a pipetting error, but it seems like it would still be amplified significantly after 40 cycles.
My second question has to do with my dissociation curve. 
Now I know that I need to optimize my reaction conditions because the baselines are really jumpy, but does that look right? My no template control is the orange triangle and my positive control is the blue "x". The higher fluorescent peaks correspond to transcript levels root tissue and the first 3 internodes while the lower fluorescent peaks correspond to levels in the last 3 internodes and my controls.
My school just opened a new lab and ordered a bunch of fancy equipment that nobody seems to know how to use, so it is up to me to figure these things out. Any help would be greatly appreciated. I can post additional screenshots of my plate setup/results upon request.
Thanks,
Hank
Also, I should mention that I performed traditional RT-PCR prior to real-time with a similar transcript level disparity between the 3rd and 4th nodes, so I know it isn't a problem with the setup.
I have looked a bit at your results and even though it doesnt please me to say so, it looks like you have very little (if any) amplification. The give away of such a problem is the jumpy baseline. This is though not a direct effect of your reaction, but rather an effect of the Mx3000 software. The software automatically corrects the values on the y-axis. In your case your fluo-values (-Rn (t)) goes from 0 to 100, while they (had you had a significant amplification) should be from 0 to 3000-5000. With a y-axis going from 0-3000 your jumpy baseline would appear flat.
Try running the results of you realtime PCR on a standard agarose gel (ethidium bromide). If you do not have a band of the right size in the gel, you need to optimise your reaction until you get dissiciation curve values in the 2000-range. At least for your positive controle.
Good luck
Anders
That makes sense, I think. But why would my amplification plot show amplification then? Maybe it's just in the way I'm choosing to represent the fluorescence. Here's my amplification plot:
I'll try running my reactions on a gel and see what happens. Thank you.
-Hank
I'll try running my reactions on a gel and see what happens. Thank you.
-Hank
Your amplifications seems fine.
Your dissociation curve on the other hand is crap (sorry - just calling a spade for a spade.)
Your dissociation curve depicters -Rn`(T)) against temperature. Is that an experiment with a reference dye? Because in that case you might be showing the dissociation curve of the reference dye rather than the SYBR green. Try engaging other "fluorescence" and see what happens.
This is though a wildcard solution.
Have more good luck.
Anders
I did use ROX as a reference dye, but I only enabled FAM in the analysis of the dissociation curve. I'll give Stratagene a call tomorrow and see if they can help me out. I really appreciated your help though, thanks.
-Hank
I wonder if you got your problem solved. I have got another strange result. The amplification curve shows amplification, and samples reaching a plateau. The melting curve shows a peak for the positive control and not for the negative control, but no band appears in an EtBr agarose gel. Hm.
I haven't optimized the reaction properly yet, and I have a high baseline for any sample with any RNA. Probably nonspecifics.
Well, I just ran my products out on a gel and I definitely have amplification and my product size is correct. I'm going to call Stratagene regarding the jumpy dissociation curve baseline.