Normalisation

DrDTonge · 06-03-2011, 12:54 AM

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

DrDTonge · 06-06-2011, 03:44 AM

Sorry guys...any thoughts?

pbluescript · 06-06-2011, 04:31 AM

I don't do small RNA sequencing, but generally, I do put in the same amount of input. However, as the actual sequencing requires such a tiny amount of library, you usually just sample a small aliquot from your finished library. If you are worried about some libraries failing, then I would start with more.

DrDTonge · 06-06-2011, 07:13 AM

Thanks for your response.

Given the large variation between samples (this is expected as they are from different disease stages) I am going to start with a standard volume of RNA (3ul) and then normalise by adding a fixed concentration of cDNA library into the sequencing reaction.

Best wishes,

D

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06-03-2011, 12:54 AM	#1
DrDTonge Member Location: MRC Join Date: May 2011 Posts: 23	Normalisation 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

06-06-2011, 03:44 AM	#2
DrDTonge Member Location: MRC Join Date: May 2011 Posts: 23	Sorry guys...any thoughts?

06-06-2011, 04:31 AM	#3
pbluescript Senior Member Location: Boston Join Date: Nov 2009 Posts: 224	I don't do small RNA sequencing, but generally, I do put in the same amount of input. However, as the actual sequencing requires such a tiny amount of library, you usually just sample a small aliquot from your finished library. If you are worried about some libraries failing, then I would start with more.

06-06-2011, 07:13 AM	#4
DrDTonge Member Location: MRC Join Date: May 2011 Posts: 23	Thanks for your response. Given the large variation between samples (this is expected as they are from different disease stages) I am going to start with a standard volume of RNA (3ul) and then normalise by adding a fixed concentration of cDNA library into the sequencing reaction. Best wishes, D