21
ISOLATE
RNA Kits
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4. Determination of rna yield, purity and integrity
The yield of total RNA may be determined spectrophotometrically at 260nm, whereby
1 unit of absorbance (A
260
) = 40µg of single stranded RNA/ml. The purity can also be
determined spectrophotometrically from the ratio of the relative absorbances at 260
and 280nm. Good quality RNA will have a A
260
/A
280
ratio in the range of 1.7 to 2.1.
The most common procedure for determining RNA integrity is running 2-4µg of a
total RNA sample on an agarose denaturing gel. The RNA may be visualized by EtBr
staining, which reveals the ribosomal RNA bands. These bands can vary depending on
the organism the RNA was extracted from (see table on page 21). In general, for good
quality RNA the bands should be distinct, with no smearing underneath them and the
28S band (larger) should be approximately twice as intense as the 18S band.
5. optional Dnase Digestion
Generally, DNase digestion is not required since ISOLATE technology efficiently
removes most of the DNA without DNase treatment. However, further DNA removal
may be necessary for certain RNA applications that are sensitive to very small amounts
of DNA (e.g., real-time RT-PCR analysis with a low-abundant target), or samples very
rich in DNA such as spleen tissue.
DNase is added to the filtrate of the R1 column and incubated 15min at 20-30°C before
the addition of ethanol 70%. Although the DNase will be removed by the next extraction
steps, a final heat inactivation (10min at 75°C) can be performed on the final eluate, to
remove any residual DNase activity.
