2 replies to this topic

[Baars01]

Hi,

In order to have equal amounts of RNA in my reverse transcription reaction (destined for qPCR) I equalise the RNA concentration of all samples. My question is, should this be done before or after the pre-RT DNA digestion step?

Thanks

[gfischer]

Baars01, on Mar 3 2010, 08:11 AM, said:

Hi,

In order to have equal amounts of RNA in my reverse transcription reaction (destined for qPCR) I equalise the RNA concentration of all samples. My question is, should this be done before or after the pre-RT DNA digestion step?

Thanks

It depends on how you measure [RNA].  If you're using a spec, then yes, because UV absorbance will measure all nucleic acids together.  If you're using an RNA specific method, like Ribogreen from Invitrogen, then you could probably measure before DNAse treatment.

[Baars01]

gfischer, on Mar 3 2010, 09:05 AM, said:

Baars01, on Mar 3 2010, 08:11 AM, said:

Hi,

In order to have equal amounts of RNA in my reverse transcription reaction (destined for qPCR) I equalise the RNA concentration of all samples. My question is, should this be done before or after the pre-RT DNA digestion step?

Thanks

It depends on how you measure [RNA].  If you're using a spec, then yes, because UV absorbance will measure all nucleic acids together.  If you're using an RNA specific method, like Ribogreen from Invitrogen, then you could probably measure before DNAse treatment.

Thanks for you reply

I have noticed a drop in nucleic acid concentration after using the Turbo DNA-free kit from Ambion and measuring with Nanodrop. The volume of the samples increase slightly, but it does not always account for the drop in concentration. Any ideas on this?

Edited by Baars01, 04 March 2010 - 08:02 AM.