Quantitative Genomics Core Laboratory 10/06/08
DNase I Treatment Protocol
1) Determine the concentration (A260) for each total RNA sample (not mRNA!!)
2) Dilute each sample to 30 or 55 ng/µl (for ≈50 or 100 ng RNA/reaction ﬁnal)
3) If RNA is limiting, the amount/well can be as low as 10 - 40 ng. The RNA
concentration is a function of the abundance of the transcripts being measured in the
sample. Since that information is the goal of the experiment, put as much RNA
concentration as possible into each sample if the above amounts are too high.
4) If necessary, dilute your commercial RNase-free DNase I enzyme to 1 U/µl using
nuclease-free PCR buffer and H2O. The buffer that comes with Taq polymerase will
work. Note that some commercial enzyme preparations are at 1 U/µl.
5) The robot will add 2 µl of RNA sample/well; 4-wells per assay. The robots need more
volume than 8 µl/assay so plan on 12 µl total RNA/assay, minimum, when
determining the volume of sample to treat with DNase I.
6) It is critical that you do not give us too little volume to perform the number of assays
requested. Always error on more RNA sample volume, not less. We can return the
RNA samples back to you.
7) Add up to 4 µl of 1 U/µl DNase I to a solution of total RNA in 250 µl of 1 mM MgCl2.
For example: 210 µl total 30 - 55 ng/µl RNA + 4 µl DNase I + 5 µl 50 mM nuclease-
free MgCl2 + 31 µl nuclease-free H2O. For more/less volume or more/less RNA
concentration, scale the amount of DNase I proportionally.
8) Incubate the tubes at 37˚C for 30 minutes, followed by 75˚C for 10 minutes then on
ice immediately. It is critical the 75˚C step is 10 minutes only. Store the RNA at -80˚C.
9) Remember to use the nuclease-free 1.5 ml screw-cap tubes we need for the robots
(ISC Bioexpress cat# C-3295-2) for submission of the ﬁnal samples. The DNase I
treatment can be performed in these tubes.
10) Label each sample with a number only, e.g., 1, 2, 3, … We do not need to know the
identity of each sample for transcript quantiﬁcation.
11) For critical RNA samples, consider using Ambionʼs (ABI) Turbo DNase (AM1906).
The enzyme is more active than DNase I and requires no extraction or heating to
remove the enzyme from the RNA samples.
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