Dear all,
Apologies if this is a naive question however I'm just wondering at what stage people implement normalisation steps into their library preparation.
As a bit of background, I have 30 samples which I wish to run small RNA sequencing (my interest is miRNAs) and then compare the relative expression of these.
Do people start each reaction with the same quantity of small RNA (as determined by the small RNA chip) and then control for cDNA yield OR input a fixed volume of RNA and then control for cDNA yield?
I would always usually control at every single stage, however, some samples have much lower small RNA concs so I don't want to risk inputting the lowest amount and risk the libraries failling.
Your help would be much appreciated,
D
Apologies if this is a naive question however I'm just wondering at what stage people implement normalisation steps into their library preparation.
As a bit of background, I have 30 samples which I wish to run small RNA sequencing (my interest is miRNAs) and then compare the relative expression of these.
Do people start each reaction with the same quantity of small RNA (as determined by the small RNA chip) and then control for cDNA yield OR input a fixed volume of RNA and then control for cDNA yield?
I would always usually control at every single stage, however, some samples have much lower small RNA concs so I don't want to risk inputting the lowest amount and risk the libraries failling.
Your help would be much appreciated,
D
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