I am comparing miRNA expression levels in 3 different groups but I am low on money and time. I have to get some preliminary results to get the actual research going so I decided to pool my samples and get the results for 372 miRNAs and then if any of them shows prominent differential expression, I will investigate the expression levels of those candidate miRNAs in larger groups. But I can't decide how to group my samples for pooling. Should I pick 3 individual samples for each of the 3 groups (9 samples in total) or should I extract the total RNA for each sample for each group, make 3 pools of RNA and then just perform triplicates for each pool? The time and money spent in both situations is going to be the same. I think the second method is more appropriate yet I fear it might produce some deflated p-values, resulting in false discovery.
1 Answer
Should I pick 3 individual samples for each of the 3 groups (9 samples in total) or should I extract the total RNA for each sample for each group, make 3 pools of RNA and then just perform triplicates for each pool?
Depends on the nature of your samples. In general, pooling would average out the expression of different individuals. Pooling is generally done when you have low RNA yield per individual sample. If that is not the case then you should sample multiple individuals per group (three should be fine; more is better). This will let you know inter-individual/sample variation within a group. You can in turn set up three technical replicates for the qPCR to measure technical variation. So, in total, you would have 27 PCR reactions. This would not be very costly (unless you are using TaqMan like assay. Even then, sometimes working with more number of samples allows you to save the amount of reagent used per sample.