Hi Ashleigh,

To answer your questions: GAPDH - it doesn't really come up "late". The Ct very much depends on the amount of RNA you used for the RT reaction and your dilution before the qPCR. 28 for a housekeeping gene is absolutely fine, but if you can, use at least one or two others and normalise to the geometrical mean of all housekeeping genes. I actually prefer HK genes that don't come up very early, because you might loose differences in samples due to the very high expression (just try e.g. 18S and you'll see what I mean).

Why do you think your knockdown didn't work? If your GOI comes up 31 in the controls and 2 CT cycles later in the KD samples, you would have a 4 fold knockdown, assuming same Ct-cylce for the HK gene. You said your "GOI coming up later in the control sample" - this is different to what you wrote before (3rd sentence) about the Ct-cylces. Hope I could help a little,

Andreas

I would check the expiry date of the reagents again, if you haven't done so already. It seems that you have a small decrease of your target mRNA anyway, right? Perhaps you should increase the amount of siRNA. Concerning the housekeeping gene: have you tried other targets instead of GAPDH? I would go for beta actin and/or HPRT.

do you perform serial dilution to determine appropriate cDNA and primer concentration?

If your HK gene (GAPDH) is the same in all samples, surely this is what you would expect. The fact that this appears at cq of 28 should not really matter. This will be an indication of the amount present in your samples. The cq value will change depending the values you have set to determine the threshold in the software that you used for analysis.

You infer that you were using a different machine earlier ?

"They had a biorad machine and did 2 step cDNA synthesis/qPCR"

Are you using a different machine now ? is sample model/brand etc

"and why is GOI coming up later in the control sample? "

Later than what ?

You state that the GOI value is 31 in control and 33 in test sample. This would suggest that this is more of your GOI in your control than your test sample. If your are trying knock down the expression of your GOI , is this not what you would expect ?

Are you using relative of absolute quantification ?

No conclusion can be made for your GOI because Gapdh value is very low. Try to increase the cDNA concentration/well and see if you increase the Gapdh expression.

Hi Ashleigh,

To answer your questions: GAPDH - it doesn't really come up "late". The Ct very much depends on the amount of RNA you used for the RT reaction and your dilution before the qPCR. 28 for a housekeeping gene is absolutely fine, but if you can, use at least one or two others and normalise to the geometrical mean of all housekeeping genes. I actually prefer HK genes that don't come up very early, because you might loose differences in samples due to the very high expression (just try e.g. 18S and you'll see what I mean).

Why do you think your knockdown didn't work? If your GOI comes up 31 in the controls and 2 CT cycles later in the KD samples, you would have a 4 fold knockdown, assuming same Ct-cylce for the HK gene. You said your "GOI coming up later in the control sample" - this is different to what you wrote before (3rd sentence) about the Ct-cylces. Hope I could help a little,

Andreas

Hi Ashleigh,

As Andrew and Andreas have mentioned your Ct values is absolutely fine for both your GAPDH and GOI.

You mentioned you was working elsewhere before Christmas and you did not have such issues. You must compare your old results and try to find what may have influence this new results. There may be lots of issues, such as:

Are you using the same cell lines and conditions as before?

Using the same kits for RNA extraction and cDNA synthesis, RNA concentration, qPCR mastermix, primer/probe concentrations?

You've already mentioned about using different PCR platforms, was the PCR cycle similar?

Also check what Gorji and David said, if you did that at your old place where you were working before, it may not be the same now.

Hope that helps,

Mano

It is always suggested to select an hk gene with a ct close to your gene of interest, in that case gapdh seems good enough.

I agree with Andreas. It looks as if you have about 4 fold down-regulation of GOI, and that your GOI is a low expression gene. You should include additional referencegenes, though, especially since these aren't enormous fold changes you're seeing.

If you have not already done so, you should also verify that your primer efficiencies are as you expect them using a standard curve for both GAPDH and GOI. If these Cqs really are later than you expect them, it could be due to poor primer efficiencies.

If your question is that you see a Cq shift performing the same experiment with the same materials between the two different labs/qPCR machines, it may also be due to differences in how Cqs are assigned. The BioRad CFX software offers two methods: a regression method and a threshold method. The former gives consistently earlier Cqs than the latter for the same experiment, and than threshold methods from many other machines, including the Roche Lightcycler 480 and the ABI 7500.