Is it worthy to pool all the samples together belong to a same group, but originated from different animals of a same species, to save time and resources (reagents)?
For example; From my negative control (untreated) group I have 5 mice and target organ is their kidneys. Now, after RNA extractions and reverse transcriptions of each animal organ (kidney) separately, can I pool all the 5 cDNA together, then measure it and dilute it and finally run this as a one sample in triplicates for the untreated group on HKG and all the GOIs included in my experiment?
Exactly the same I want to do with the other groups of various treatments for my study design.
Would it have any impact or harm on data or at the calculations and is it wise enough to do so? (especially, if I have less time and resources)
Any suggestions!!!!!
Thanks....
Hi Jawwad,
I cannot recommend this strategy. Essentially you will not get any information about the biologic variability in your groups. This would reflect an experiment with n=1 in each group and would be not sufficient to draw valid conclusions. To save money you may check if you can reduce reaction volumes, the amount of enzymes or you can analyze a subset of samples to decide which treatments show the strongest effects to concentrate on these. If you qPCR assays are stable and the changes in gene expression are large you may also switch to technical duplicates instead of triplicates.
Good luck
Norman
Hi Jawwad,
I cannot recommend this strategy. Essentially you will not get any information about the biologic variability in your groups. This would reflect an experiment with n=1 in each group and would be not sufficient to draw valid conclusions. To save money you may check if you can reduce reaction volumes, the amount of enzymes or you can analyze a subset of samples to decide which treatments show the strongest effects to concentrate on these. If you qPCR assays are stable and the changes in gene expression are large you may also switch to technical duplicates instead of triplicates.
Good luck
Norman
Hi Jawwad,
a good idea to gain time and money, but a very big for data analysis since it will be impossible for you to distinguish samples. On the other hand, if you want to compare only groups, respecting of course the states of the art for qPCR assays, it could give you, if sufficiently sensible your assay is, an idea on what you have in both groups.
fred
I will never recommend pooling similar samples (as they represent biological variables). The more the samples, better it is for statistical analysis and more confident you can be of your result. We perform qPCR in 384-well plates and the reaction volume is only 10ul (barely 3ul of cDNA/well is needed). If you are good at pipetting, go for duplicates than triplicates. I hope these steps may help you save money as well as time and finally you can have enough data points to perform calculations and statistical analysis.
I agree with Rashmi and Fred. But it also depends on your experiment. If I understand your question, are you asking to pool several grps of five biological samples as 1 biological sample (and have 2 other samples of five pools each as biological replicates) and have triplicates of same sample as technical replicates? Or are you asking to pool samples and from the same have triplicates of biological samples in which case they are actually technical replicates?
First, I would never use the delta delta method. This method is prone to many error. The most important one is the assumption that the efficiency of alle reactions is "2". This is seldomly the case. Even a slight difference in efficiency, e.g. 5% (which occurs easily) causes a considerable deviation in Cq within an experiment.
Second, if you want to pool your results, you must first check the efficiencies of the individual amplification reactions (which is only possibler when you use LinRegPCR). And if you use LinRegPCR, I would advice you to calculate expression with the N0. The N0 corrects for different efficiencies and applies for all samples with the same target. Is done so, you can combine results with a a dedicated program Factor Correction: removal of between session .... Both programs can be downloade from HFRC downloads
Hey Elizabeth, May I have your email. I have had similar concerns about the delta-delta method. Please may I have your email so we can discuss these methods further? My email is [email protected]
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