Protocols
To view these protocols, you will need Adobe Acrobat
reader which can be downloaded for free from the 'Other
Documents' area.
Spotted arrays
Target (RNA) Labeling
There are a number of basic methods for labeling
RNA prior to hybridisation to microarrays. Below are
listed a few of the most commonly used methods
- Direct incorporation:
Perhaps the most widely used approach for labeling
target. Fluorescent label is incorporated into first
strand cDNA during reverse transcription of the RNA.
This method is robust, but requires a relatively large
amount (25-100 µg) of total RNA to produce a
strong hybridisation signal. We routinely use the
method given here for labeling target for QC testing
of arrays.
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HERE TO DOWNLOAD
-
Aminoallyl
(indirect) incorporation: An increasingly
popular technique. We have little first hand experience
of this method but some of our collaborators swear
by it. Reports suggest that you require less starting
RNA and that it is also a robust method .
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HERE TO DOWNLOAD
Labeling approaches for samples in which target
RNA is limiting (50 ng-5 µg)
-
Strand-switching
PCR: Amplification
method involving first strand cDNA synthesis and
then PCR amplification. This approach differs from
other methods in that first strand synthesis is
accomplished using a strand-switching oligonucleotide
which ensures that full-length cDNA species are
produced.
Amplification of 50 ng of target RNA is achievable.
Clontech have commercialised this technology
(SMART™).
This protocol is recommended for use with HGMP-RC
fabricated arrays.
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HERE TO DOWNLOAD PROTOCOL
- Genispheres DNA dendrimer
probes: Specifically designed for use in situations
where target RNA is limiting (1-5 µg required).
Use of a 'heeled' primer for reverse transcription
allows subsequent capture of a large DNA dendrimer
molecule loaded with label before or after hybridisation
of the target. Full details of the technology and
protocols are available on Genisphere's web site.
Some collaborators are producing nice results using
this approach.
CLICK HERE TO ACCESS THE GENISPHERE WEB SITE
- In Vitro Transcription
method: An approach for amplification of down
to 100 ng RNA (albeit with multiple rounds of IVT
potentially being required). Ambion have commercialised
this technology.
CLICK HERE TO ACCESS
THE AMBION WEBSITE
- Single Primer Amplification:
An approach for amplification of down to 30
ng RNA. The protocol involves
first strand synthesis primed by a 5' modified oligo(dT)
primer, second strand synthesis and then Taq
polymerase amplification primed with an oligonucleotide
equivalent to the heel of the oligo(dT) primer.
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HERE TO DOWNLOAD THE SPA PUBLICATION
Microarray hybridisation and washing protocol –
manual
Microarray hybridisation and washing protocol –
automated (using the Amersham Lucidea SlidePro) -
In our
hands automated hybridisation and washing
improves uniformity of hybridisation and reduces background.
Although protocols are dependent on type of array (cDNA
vs oligonucleotide probes) and type of slide,
the following downloadable protocols are the two which
we routinely use.
The HGMP-RC microarray group use and recommend Strand-switching
PCR for labelling and the ASP for automated hybridisation
and washing.
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