mRNA quantification - spectrophotometric method (Sep/03/2004 )

Hi,
I work on mRNA extracts from fruits and I need quantify it to synthetise cDNA, but i just have a little amount of mRNA (100 µl). Do you think that i can do a dilution (maybe 2 µl of mRNA diluted in 100 µl DEPC water) ? or is it too low ? Does anybody know an other method to quantify mRNA without damage it, let me know ? Thanks in advance.

I don't know how much RNA you get from your samples, but usually I read RNA concentrations at 1:50 dilutions so 2ul in 100 should work. I wouldnt try to quantitate RNA in Depc water, I think it might affect the reading. I would use autoclaved Milli-Q filtered water, The best thing to do is to autoclave your high quality water in ~1mL aliquots in 1.5mL tubes, open each tube once or twice then throw it out. Also try to find access to one of the new eppendorf or Biorad specs, they sell sterile cuvettes so that you could use your diluted RNA for an experiment after you read it if it is at the correct concentration.

The quantification of RNA in DEPC water is OK if you reference the spec with the same water you made the RNA dilution in.

I usually dilute 1:200 (3.5 ul into 696.5 ul, depending on what is the minimal solution required by the cuvtte) in water for spectrophotometry.

I usually dilute 1:70 to quantify the mRNA, but it is better to quantify it with duplication for the great variation can always lead to wrong results.

Good lucky!!

DEPC water tends to have a low pH, in turn low pH affects the ratio 260/280 of your sample, i would recomend using a solution with pH close to 7 or 8 like TE for you spec readings if you want to know the ratio. Otherwise it is only useful to use DEPC water for quantitation, not for the quality of your RNA.
Potzu