Best protocols for measuring RNA concentration

[mountain]

Do you have any good protocols for measuring the concentration of RNA? The ones i have they seem to be very confused.


Thanks.

 

[iansmith]

I usually used UV spectrometry. I put 10 uL of RNA into 990 uL of RNase free water. My blank is 5 uL of the suspension liquid for your RNA into 990 uL of RNase free water.

You measure the Absorbance at 260 nm, 280 nm and 320 nm. 260 is for nucleic acid, 280 is for protein and 320 is your background.

(Net A260 - Net A320 ) x RNA extinction coefficient (40 for RNA) x dilution factor (1000/10) = RNA concentration in ug/mL

Purity of sample = Net A260/Net A280. for pure RNA you should have a ratio of 1.7 to 2.0

If you have problem with the reading, it might be due to the fact that your RNA is not well mixed in the dilutent. You can heat up the sample at 55C for 10 minutes and then take the reading. Also make sure your DNA is well resupended in the sample you will used for measuring the concentration. Vortex the sample for about 5 sec prior to taking the reading.

Also used the same cuvette for your blank and sample. Make sure the cuvette is cleaned between sample. You should rince with water about 20 times.

 

[Moonbear]

If you have problem with the reading, it might be due to the fact that your RNA is not well mixed in the dilutent. You can heat up the sample at 55C for 10 minutes and then take the reading. Also make sure your DNA is well resupended in the sample you will used for measuring the concentration. Vortex the sample for about 5 sec prior to taking the reading.

If you have problems with the readings, sometimes it's also worth trying another spectrophotometer! I was going nuts once trying to figure out why I was having such a problem with purity/yield of my samples, and then someone suggested I try the spectrophotometer in another lab. Sure enough, there was a problem with the equipment, not my samples.