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78
RNeasy Mini Handbook 10/2019
For determination of RNA concentration, however, we recommend dilution of the sample in a
buffer with neutral pH since the relationship between absorbance and concentration (
A
260
reading of 1 = 44 µg/ml RNA) is based on an extinction coefficient calculated for RNA at
neutral pH (see “Spectrophotometric quantification of RNA”, page 76).
DNA contamination
No currently available purification method can guarantee that RNA is completely free of DNA,
even when it is not visible on an agarose gel. While RNeasy Kits will remove the vast majority of
cellular DNA, trace amounts may still remain, depending on the amount and nature of the sample.
For analysis of very low abundance targets, any interference by residual DNA contamination
can be detected by performing real-time RT-PCR control experiments in which no reverse
transcriptase is added prior to the PCR step.
To prevent any interference by DNA in real-time RT-PCR applications, such as with ABI PRISM
®
and LightCycler instruments, we recommend designing primers that anneal at intron splice
junctions so that genomic DNA will not be amplified. QuantiTect Assays from QIAGEN are
designed for real-time RT-PCR analysis of RNA sequences (without detection of genomic DNA)
where possible. For real-time RT-PCR assays where amplification of genomic DNA cannot be
avoided, we recommend using the QuantiTect Reverse Transcription Kit for reverse
transcription. The kit integrates fast cDNA synthesis with rapid removal of genomic DNA
contamination (see ordering information, page 91).
For other sensitive applications, DNase digestion of the purified RNA with RNAse-free DNase
is recommended. A protocol for optional on-column DNase digestion using the RNAse-Free
DNase Set is provided in Appendix D (page 82). The DNase is efficiently washed away in
subsequent wash steps. Alternatively, after the RNeasy procedure, the RNA eluate can be
treated with DNase. The RNA can then be repurified according to the RNA cleanup protocol
(page 67), or after heat inactivation of the DNase, the RNA can be used directly in
downstream applications.
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