analyzing from multiple plates - (Feb/18/2008 )

Hi
I'm doing realtime PCR using sybrgreen chemistry. i need to analyze 7 genes in 11 species and three reference genes. in each species i need to compare two stages. i have designed species specific primers. for each gene i cannot have all the samples in single plate. so what precautions i should take when i start such an experiment in order to be able to compare the ct values across different plates.
thanks
murali

---

you should prepare some kind of calibrator sample. for example from pooled cDNA of your experimental samples. this calibrator is measured on every plate for every target gene. for analysis, you can calculate the expression levels of your target genes in your samples in relation to the calibrator on the respective plate.

---

thanks for the quick reply. another question is will it be fine if i run the control genes and target genes on two different plates. what kind of calibrator might be required in order to be able to use relative quantitation method? or will it be fine if i run the control genes on one plate and target genes on the second plate and compare them?
thanks once again
murali

---

You can run different genes on different plates, but you MUST have all samples and controls on the same plate. You don't need to have a calibrator, because you're comparing relative differences between your samples and controls, and these are run together.

---

I would put all of the genes with the three ref genes for one species from the two stages at the same plate and then do the same for the rest of the species. Also there was a software which can normalise for interrun variation it is called Factor Correction for between session variation (Ruijter 2006).

---

How exactly are you setting the experiment (number of replicates) and what's your plate size (96?)? And most importantly, what do you want to compare with what? Those two stages to each other only or all the species and stages together?
If you compare within one species only, you can do it the way yaourt suggested.

---

I have three replications for each sample and for the serial dilution and my plate size is 96. what I'm doing is I'm trying to compare expression differences between two different stages in a given species and like this for 11 different species. I'm not sure if i can compare the stages between species but if possible, I will do it otherwise i'll just stick to only between stages within a given species. but loading all the species on same plate is not possible for me as different species have their own species specific primers and each species since I need to check efficiency of all these primers, i don't think i will be able to load them on same plate. that is why I'm trying to explore the possibilities to compare inter-plate variability. as yaourt suggested I'm going through the Ruijter 2006 manuscript. any other suggestions!!!!!!!????
thanks to u all
regards
murali

---

In that case be only sure to put both stages on a same plate, because you want to compare them, not the species between themselves.
If the primers you use are different for each species, you cannot compare them anyway.

---

hi trof, do you have any other suggestion to compare different plates as i cannot run the control genes and target genes on the same plate. at the moment i think including a calibrator is a reasonable option.
thanks
murali

---

I think I'm getting a bit lost in this.

I had more than 40 samples from two different treatments, needed to run in triplicates for 3 housekeeping and 6 target genes. 20 were to be compared together (one type of treatment). That's more than 63 for one gene (without dilutions as I know efficiencies are similar and including non-template control). This 20 from first treatment was the first "block of samples".

For first "block of samples" I simply run each gene on separate plate, then used Cts for relative quantification. No calibrator needed.
I did the same for the second "block of samples". I could compare values within block, but not between them.


Its all about what you need to compare. If you want to compare first stage to a second stage, you need to run only these two on one plate for a single gene. If not, or if you're not sure, use a calibrator and recalculate values on other plates.
This is something you need to decide, but basic rule stays the same: All samples to be compared together on the same plate for one gene. It doesn't matter if other gene(s) are run on different plates, because interrun differences would be same for all the samples on plate and will nullify in the end sum.

Hope It's clear enough.

---