RNA Expression Microarray Sample Guidelines
We expect that your samples will be supplied in the correct containers and at the
required concentration and quality (requirements are stated below). Timely
completion of your project depends on you following these guidelines.
We use the Illumina® TotalPrep™-96 RNA Amplification Kit made by Ambion / Life
Technologies (http://tools.lifetechnologies.com/content/sfs/manuals/4397949E.pdf)
to produce labelled cRNA for hybridization and we advise you to refer to this
information when preparing your samples.
Study Design Considerations
Before we accept samples for analysis we will need to consult and agree with you on
the design of your experiment, including issues such as technical and biological
replication, randomization and the possibility of confounding of experimental groups
or treatments with placement on the Illumina slides in the experiment. Where it is
experimentally practical, please provide at least 3 and preferably 4 or 5 independent
(biological) replicates per condition or experimental group under study.
Sample Quantity
The current protocol requires total RNA with all samples normalized to a single
concentration of 25-100ng/ul as measured by NanoDrop. Please provide two
aliquots of each sample, in separate plates: one of 12-15μl for sample processing
and another one of 3-5μl for QC. If you prefer to undertake your own QC then the
second aliquot is not required, however we cannot guarantee labelling efficiency and
the overall performance of the experiment and we will need to check that you
understand the consequences of this option (see below).
Sample Quality
For robust results, the RNA Integrity Number (RIN) of each sample needs to be
greater than 7 and samples should have a 28S/18S ratio of greater than 1.6. The
260/280 ratio should be 1.8-2.0 and the 260/230 ratio should be close to 2.0. We
test these quantities as part of our QC process before undertaking labelling,
although you may choose to check your RNA in advance. If the results of QC tests are
unsatisfactory and we and you consequently agree not to continue with the
experiment we will normally return the remaining samples to you and charge only
for these QC steps; otherwise a project can be run at your risk.
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If you opt not to send us the extra sample aliquots to make use of our QC service, we
advise you to send us evidence of your QC process (e.g. Bioanalyzer files and further
sample information as below) so that we can evaluate the quality of your RNA
samples.
Sample Plating and Shipping
Please arrange RNAs in a 96 well 0.2ml semi-skirted plate (Thermofisher, ABgene, ref
AB-0900) in columns (A1, B1, … etc) in the order that you wish them to be run.
Please put the aliquots for processing and QC on a different plates, clearly label the
plates with your name and date, and seal plate using adhesive seals (Thermofisher
ABgene ref AB-0558 or similar) capable of withstanding changing temperatures,
ensuring each well is properly sealed. Pack the plates in a container or bag with
sufficient dry ice to stay frozen during transport.
Please email an electronic sample sheet (MS Excel) with as much sample information
as possible (Sample ID, Sample Experimental Group, Concentration ng/uL, Ratio
260/280, 260/230, RIN value, Test Sample Volume, QC Sample Volume, Slide
Number to which the sample is assigned) and a brief description of your project.
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