minimum replicate for qpcr? - (Apr/13/2011 )
What is the minimum number of "biological replicate" and "number of reaction replicate for each biological replicate" that is required for qPCR? 2 biological replicates, each 2/3 reaction replicates will be enough or not?
I'm not sure if it's required, but usualy 3 replicates (both biological and reaction) are a good choice, because if one of the replicates flies off you can discard it and use remaining two. If you have only two replicates and they differ you can't tell which one is right and which one is off.
But when your two biological replicates show the same results, maybe you don't need to do the third.
Trof on Thu Apr 14 06:06:01 2011 said:
I'm not sure if it's required, but usualy 3 replicates (both biological and reaction) are a good choice, because if one of the replicates flies off you can discard it and use remaining two. If you have only two replicates and they differ you can't tell which one is right and which one is off.
But when your two biological replicates show the same results, maybe you don't need to do the third.
I see... Is this how replicates work? Sometimes in experiment, one replicate may deviate far from the other two. So should we discard that one only and get the mean of the other 2, or repeat another replicate to replace it or use the original 3 replicates? Because doesn't it sound like manipulating the result if we discard the one with odd/non-desired result?
Well, intention is to discard the odd, never the undesired. When in group of three one is far from other two, then it's more likely that something went wrong there, you may actually like the wrong one better, but you still should discard it. Keeping it with other two and making mean of them all will shift the value and bring more error to your calculations. If you ask whether you should repeat it, well ideally yes. But that could mean repeat whole bunch of samples, that are compared with the affected one. But if your other two replicates are close to each other I don't see much point in that, apart from having triplicates "to the letter". Sometimes mean of duplicates (with low variability) is sufficient so making a reliable duplicate from a triplicate wouldn't actually matter.
Trof on Thu Apr 14 11:05:27 2011 said:
Well, intention is to discard the odd, never the undesired. When in group of three one is far from other two, then it's more likely that something went wrong there, you may actually like the wrong one better, but you still should discard it. Keeping it with other two and making mean of them all will shift the value and bring more error to your calculations. If you ask whether you should repeat it, well ideally yes. But that could mean repeat whole bunch of samples, that are compared with the affected one. But if your other two replicates are close to each other I don't see much point in that, apart from having triplicates "to the letter". Sometimes mean of duplicates (with low variability) is sufficient so making a reliable duplicate from a triplicate wouldn't actually matter.
Now i got it! Thanks! Another question:
I was told that ref gene must be run concurrently with target gene reaction because efficiency of every reaction each time is different. This means that both ref gene and target genes must have same Ta. Is this true? I have 8 target genes and 1 ref gene and i found it difficult to make their Ta close. They ranged from 50-55C.
You don't need to run all genes on one plate if you're doing relative quantification. What you must on the other hand is to run all samples on one plate/run. Because you compare the samples with each other. You don't compare genes. Any difference in efficiency between genes would be canceled out.
So no, you don't need to have same Ta.
I agree with trof, though all the time if that appears and you keep doing it its not a good practice... you may have to improve on your skills... also such samples are generally termed out of trend (OOT)
Trof on Tue Apr 19 08:49:49 2011 said:
You don't need to run all genes on one plate if you're doing relative quantification. What you must on the other hand is to run all samples on one plate/run. Because you compare the samples with each other. You don't compare genes. Any difference in efficiency between genes would be canceled out.
So no, you don't need to have same Ta.
That means when running all samples in the same plate, i dont have to include a ref gene in it? I was told that "they must be put together (ref and target gene) in the same run because at the end we will compare the conc of target with conc of ref. Since each run has diff efficiency, it is not comparable."
hianghao on Thu Apr 21 00:12:08 2011 said:
That means when running all samples in the same plate, i dont have to include a ref gene in it? I was told that "they must be put together (ref and target gene) in the same run because at the end we will compare the conc of target with conc of ref. Since each run has diff efficiency, it is not comparable."
Are you doing delta-delta Ct (or efficiency corrected Pfaffl version) or something else? Are you doing relative quantification? If yes, then you compare samples not genes, as I wrote, you can have each gene on different plate.
Trof on Thu Apr 21 04:48:34 2011 said:
hianghao on Thu Apr 21 00:12:08 2011 said:
That means when running all samples in the same plate, i dont have to include a ref gene in it? I was told that "they must be put together (ref and target gene) in the same run because at the end we will compare the conc of target with conc of ref. Since each run has diff efficiency, it is not comparable."
Are you doing delta-delta Ct (or efficiency corrected Pfaffl version) or something else? Are you doing relative quantification? If yes, then you compare samples not genes, as I wrote, you can have each gene on different plate.
i m doing relative quantification. Not sure which method to use as from what i read from qiagen qpcr manual, it mentions that the calculation method depends on whether pcr efficiencies are similar. If they are, i'll use delta-delta ct. Else, i'll convert the ct of treatment sample and convert it to conc based on standard curve, normalise target gene, then compare the expression with that of calibrator sample (dn't know the methods name).
I am studying expression of diff genes after 9 different types of treatments with diff xenobiotics and compare it with unexposed as calibrator sample. So it means that i had to put 3 (biological reps)X 3 (rxn reps) x 10 (treatment + calibrator) in each plate? What if i got more treatment and they can't fit into the plate? Do i have to do no RTase ctrl if i'd conducted DNAse treatment?
Must the conditions (eg. primer conc and template conc, dilution rate and conc used to prepare standard curve) be the same for each target gene?