I have problem about the number of control samples in each of the steps of stem loop qRT.

In the 1st step (Stem-loop RT), 2 types of controls are required [no RT control and no RNA control]. I am working with 3 miRNAs and 2 different RNAs [tRNA]. So the total samples I should have are 6, without the controls. How many control treatments should I have in this step?

In the 2nd step [end point PCR], there is a 'no RT product control', so how many samples for this step?

The problem is that with this much number of samples, I can't run the qPCR [which can take the maximum 96 number of samples], while my samples might be more than 100 (according to the calculations). So what should I do?

Another question is about the 5S control that is usually used in the qPCR to analyse the results. Does that also accounts in the steps or not?

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