Dear Marcelo

1. If you are going to use qBase(Plus) for data-analysis, there is no need to use the RQ setup, so AQ is perfect.
2a. In AQ setup, you don't attribute a label to the genes; the lables NTC, UNKN, STD are for your samples.
3. No! You only need to analyze the reference genes once. You can analyse 5 genes per plate, so you need 8 plates. One of these plates contain the 2 reference genes, in addition to 3 genes of interest.
4. The choice of using a standard or not is yours. Some like it or demand it, others don't. If you have a validated assay, and you know the performance of the assay in terms of efficiency, linearity, dynamic range, limit of detection or quantification, etc., you can probably do quantification without a standard.

Regards
Jo Vandesompele

Marcelo Laia schreef:

Dear all,

We start a experiment and we planning to analyse it in qBASE.

So, we starting reading the qBASE tutorial, 1 Initial experiment.

But, we are with some doubts.

Our system is an Applied 7500, and we are using SyBrGreen and we will use 2 REF to compare relative expresion in 37 GOI in 8 samples from two groups (4 treated and 4 untreated). I would like to see relative expression between treated and untreated (we looking for the treatment effect on those genes).
[37 GOI + 2 REF] * [8 UNKN + 0 STD + 1 NTC] * 2 R = 702 wells

1. We choose Absolute or Relative in instrument's set up?
2a. If we choose Absolute quantification, we set the REF genes as UNKN or STD or NTC in instrument set up?
2b. If we choose Relative quantification, we found ENDO and Target on Task, but not NTC!
3. I will need to run ~8 plates (702/96). So, I will need to run the two REF genes in all plates? In this case, I will need to run 3 GOI and the 2 REF per plate. Or may be run the REF in the fist plate and in the next we can run 5 GOI per plate?
4. We don't planing to use standard. This is Ok or not?

Please, could you help me on how we can set up our experiment for forwarding use qBASE properly?

Thank you very much!!!

--
Marcelo Luiz de Laia
Jaboticabal - SP - Brazil

"Você vê as coisas como elas são e pergunta: por quê? Mas eu sonho com coisas que nunca foram e pergunto: por que não? " - Bernard Shaw