Mammalian RNA Quality Control for Samples to be used for Hybridization to Microarrays
Dr Ruan Elliott, Institute of Food Research, Norwich, NR4 7UA, UK.
E-mail ruan.elliott@bbsrc.ac.uk
When citing this SOP you should acknowledge both NuGO and the appropriate NuGO
partner institution that has made the SOP available. Please use a form of words such as:
We used the NuGO Standard Operating Procedure (SOP) number 90 produced bythe
Institute of Food Research. Details of the SOP are available via the web link:
http://www.nugo.org/frames.asp?actionID=39146&action=loginFromPP
Date SOP prepared:
Species specificity of SOP:
Relevant ‘omic’ technology:
Relevant section of ‘pipeline’:
26 November 2008
Mammalian
Transcriptomics,
Sample analysis
Background
Many methods and commercial kits are available for the isolation of total RNA from mammalian and
all of these are potentially acceptable although in some cases specialised protocols may be required for
efficient RNA isolation from specific refractory tissues. However, regardless of the strategy adopted, it
is vital that high quality RNA is obtained (i.e. of very high purity and integrity), particularly if the
samples are to be used for generating labelled extracts to hybridize to microarrays.
Overview
This standard operating procedure sets out the methods of analysis and the standard quality minimum
metrics advised by NuGO for RNA samples to be considered of sufficient quality for microarray
analysis. Spectrophotometry is used to determine the purity of the nucleic acid. The Agilent
Biolanalyser system (or gel electrophoresis) is used primarily to assess the RNA integrity but can also
provide indications of other possible problems such as DNA contamination.
Materials
1. Spectrophotomer suitable for accurate absorbance determinations in the range 230-280 nm.
Ideally the machine should be capable of spectrophotometric determinations with very small
volumes of sample (e.g. a Thermo Scientific Nanodrop spectrophotometer or equivalent machine).
Conventional also spectrophotometers may be used provided suitable quartz cuvettes and a
sufficient quantity of each RNA sample is available. Note that for cuvette-based systems, although
it is possible to retain the sub-sample used for analysis this is not recommended unless rigorous
precautions have been taken to ensure no contamination of the sample with RNases.
2. Agilent 2100 Bioanalyser (or equivalent) with Agilent RNA6000nano LabChips® .
Note that this is the preferred option, but where no Bioanalyser is available gel electrophoresis is
acceptable as an alternative indication of RNA integrity. In this case, the gel images should be
captured to provide a record of the analysis.
Main procedures
1. Note that repeated freeze-thawing of RNA samples is undesirable. Therefore, it is recommended
that the following quality control analyses should be performed either on the same day (preferably
the same day that the RNA samples are prepared prior to storage at -80 °C) or on separate aliquots
of stored RNA samples.
2. As for all work with RNA, standard precautions must be adopted throughout the procedures to
minimise the risk of contaminating the samples with RNases that are ubiquitous in the
environment. Necessary precautions include the use of gloves by the operator and use of
guaranteed RNAse-free plasticware (including aerosol resistant RNase-free pipette tips) and
RNase-free water (available from many commercial suppliers or by DEPC-treatment of high
purity water).
3. Spectrophotometry: the absorbance of all RNA samples should be determined at 230, 260 and 280
nm. The spectrophotometry should be performed according to the instructions of the system
manufacturer.
4. Agilent Bioanalyser analysis: aliquots of all RNA samples should processed according the system
manufacturer’s standard protocol using Agilent RNA6000nano LabChips®.
Safety
The procedures all set out above low risk although mammalian RNA should be treated as a potentially
biohazardous material.
Results analysis
The quality of all RNA samples should be determined prior to use with microarrays and the
information obtained should be recorded as part of the study metadata.
For samples to be considered of suitable quality the following parameters should be met:
1. The ratio of absorbance at 260nm/absorbance at 280 ratio should be within the range >1.8 and
<2.2
Low ratios indicate that the sample is likely to be contaminated with protein. Note also that
this ratio can be affected by the use of buffers to dilute the sample prior to analysis.
2. The ratio of absorbance at 260nm/absorbance at 230 ratio should be >1.8.
Lower ratios typically indicate contamination with organic material such as solvents used in
the isolation protocol.
3. The RIN (RNA INtegrity) scores - obtained from the Agilent Bioanalyser should be >8.O.
RIN scores range from 1-10 and are derived automicailly by the Agilent software from the
sample electropherograms using a proprietary algorithm. A RIN score of 10 indicates
completely intact RNA, whereas a RIN score of 1 indicates complete degradation. Note that
the values obtained can vary somewhat from organism to organism and tissue to tissue so this
should be considered as an advisory cutoff value. It is also important to visually inspect the
sample electropherograms and checks of what should be expected for different tissues can be
made
against
Agilent’s
RNA
integrity
database
(RINdb
available
at
http://www.chem.agilent.com/rin/_rinsearch.aspx).
References
http://www.chem.agilent.com/en-US/products/instruments/lab-on-a-chip/2100bioanalyzer/rnasolutions
http://www.chem.agilent.com/enus/Search/Library/_layouts/Agilent/PrimaryDocumentViewer.ashx?whid=37507
Schroeder A, Mueller O, Stocker S, Salowsky R, Leiber M, Gassmann M, Lightfoot S, Menzel W, Granzow M
and Ragg T. The RIN: an RNA integrity number for assigning integrity values to RNA measurements. BMC
Molecular Biology 2006, 7:3