quantity of RNA to use in RT reaction - (Jan/31/2006 )

Hi,

I have a question for you. I extract my RNA using Trireagent, and I have found that it is usually slightly degraded.
The 260/280 values are around 1.6-1.7. I read somewhere that extractions with trizol/trireagent often have these kind of values. The bands look fairly clean on the gel, but the 28 s is not twice as big as the 18S - often the 18 S is bigger.

I am doing RT using the Superscript III first strand kit from Invitrogen. Normally I would use 1 ug of RNA for this.
I wonder whether it helps to get a greater yield of quality cDNA if I assume that the actual conc. of RNA in my samples is lower than that calculated by the spec, because of the low 260/280 value, and add more than the calculated volume for 1 ug RNA to the RT reaction?

thanks

I frequently use 10 ug of total RNA, but I am unclear on the primer concentration (I see a lot of different values listed).

Could you try phenol/chloroform extraction or something similar to remove the protein impurities?

-Matt

I try to prepare as much cDNA as possible for microarray analysis and qPCR. It's not that I couldn't get away with less, but more is better.

This entails more total RNA (2.5-10 ug).

-Matt