4
Quantification and Storage of RNA
To determine the concentration and purity of RNA, measure absorbance at 260 nm and
280 nm with a spectrophotometer. One OD unit measured at 260 nm corresponds to 40
μg/mL RNA. DEPC Water is slightly acidic and can dramatically lower absorbance values.
We suggest that you dilute the sample in a buffered solution (TE) for spectrophotometric
analysis. The A
260
/A
280
ratio of pure nucleic acids is 2.0, while an A
260
/A
280
ratio of 0.6
denotes pure protein. A ratio of 1.8-2.0 corresponds to 90%-100% pure nucleic acid.
Phenol has a maximum absorbance at 270 nm and can interfere with spectrophotometric
analysis of DNA or RNA. Store RNA samples at -70°C in water. Under these conditions, RNA
is stable for more than a year.
Integrity of RNA
It is highly recommended that RNA quality be determined prior to beginning all
downstream applications. The quality of RNA can be best assessed by denaturing agarose
gel electrophoresis with ethidium bromide staining. The ribosomal RNA bands should
appear as sharp, clear bands on the gel. The 28S band should appear to be double that
of the 18S RNA band (23S and 16S if using bacteria). If the ribosomal RNA bands in any
given lane are not sharp and appear to be smeared towards the smaller sized RNA, it is
very likely that the RNA undergone degradation during the isolation, handling, or storage
procedure. Although RNA molecules less than 200 bases in length do not efficiently bind
to the HiBind® matrix, a third RNA band, the tRNA band, may be visible when a large
number of cells are used.
Quantification of RNA
