Q-PCR - Inconsistent Results - (Aug/17/2005 )

Hi everyone!

I have some problems with my Q-PCR. I am using Taqman probes to study gene expression of two genes - PLD1 and PLD2. I am using b-glucurinidase as an internal control gene.

I have been using the SIGMA RNA extraction kit -but I have also tried the Nucleospin RNA II by MACHEREY-NAGEL, just because I had seen other people in my department using it.


The problem is that even though all the times I get perfect standard curves for all three of my monitored genes, I keep getting inconsistent results. Good standard curves I believe eliminates the possibility of pipetting errors and/or bad mixing of any of my reagents, since I use triplicates per sample. Also the amplification curves when analysing the results seem to be normal, so, each time I have good amplification of all genes. Moreover, my no-amplification samples, containing everything -RT always give me exactly that -no amplification.

So, I would expect for example that PLD2 expression under the conditions I treat my RBL-2H3 (rat basophilic leukaemia) cells should always be higher than PLD1 expression. And this is kind of my control experiment trying to setup the Q-PCR. However, no two experiments give me the same results.

The triplicates I use for each sample I monitor do not have a considerable StDev in every experiment. However, after normalising them against my internal control, I get all times different numbers, which some times show that the expression of the test genes is higher than my control's and other times that it is lower, even though in every experiment they have been treated strictly under the same conditions.

At that point, I should mention that in my RT Step, I have been using 5ul of RNAt / reaction (out of the ~50ul I get from the extractions), which I have OD'd and found that it is between 1-2ug RNAt / reaction. The RT Step is performed using AMBION Retroscript Kit.

The QPCR machine is the ABI Prism 7700 SDS. The software used for analysing the raw results on the plate after the Q-PCR run has ended is the SDS 1.9.1 for Mac.

Anyone who has some idea about anything else except of pipetting errors that could cause the results to be quite different each time?

Hi George!

I am not an expert in Real Time, but as I am having similar problems maybe I can give you some ideas of what things you can check.

First of all, check if your internal control can be modified after your treatment. It has been demonstrated that some suposed housekeeping genes are not so good as internal controls. I have no idea about yours.

Secondly, which amount (in ng) of RNA do you use to do the RT ? Too much quantity can be give you worse results! I also can tell you that there are kits that perform a better retrotranscription, you can ask me if you want. However, the AMBION's one is quite good.

Finally, which amount of RNA do you use to do the Real Time? Higher amounts can give worse results!!

I don't think that the kit you use to extract the RNA is the problem.

If I have more ideas I'll tell you.

Best whishes!

Neus

Dear freinds,

Did you guys add internal calibration dye, like ROX or something else with similar function?

Best regards

good point about the ROX, also about the housekeeper..those can be flaky, especially if you are treating cells

I would like to add, it is very important to get as close as possible to exactly the same amount of template added in every repetition..."between 1 and 2 ug" on the RNA prep is pretty nebulous...

on a couple of the genes that I study, a 2X difference in starting template can affect the reaction efficiency, making the results inconsistent


and how close are your efficiencies? Assume that maybe not always the same amount of starting RNA template...this causes a chain reaction when the RT is not occuring at the same efficiency...meaning you may be adding vastly different amounts when you get all the way to the PCR plate. this can make major differences in your efficiencies

ah, to long for the carefree days when PCR was easy and you just tossed all the stuff in a tube and let it go...

good luck

Hi guys

I've been having the same problems. I set up Real-Time to measure the quantities of my mRNa against hprt. I used two RNA samples extracted from the same flask of cells, so the ratio of test/reference should be the same in each experiment.

In a single run, the amount of my gene normalised to the reference is about the same in the two RNA samples, ( SO YOU'D BELIEVE YOU'D GOT THE RIGHT ANSWER!!)
but i am getting vastly different results for these same samples in different runs (Roche, Light cycler using Qiagen SYBR green). Sometimes more test gene, somethimes less!! Arggghhh.

I'm beginning to think that it's something to do with variations in the stardard curve - the programs fits the standards run in each test to the external standard curve, but moves the intercept to do this (i just saw this on the roche website). So if your standards had a different efficency each run the values calculated could be vastly different?? Someone good at maths could work this out!! And would small differences in the standard curve make big differences to the quantification as the conc is a log scale?

Is this true - do you need to look at the intercept and slope values for each imported standard curve? (just thought of this on the way home, going to look tomorrow!!)

What does anyone else think?