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Gene array analysis has become the method of choice
for researchers who wish to compare samples for differentially expressed
genes. A typical gene array experiment involves:
- Isolating RNA from the samples to be compared
- Converting the RNA to labeled cDNA via
reverse transcription (RT) or in combination with aRNA amplification
- Hybridizing the labeled cDNA to identical membrane or glass slide
arrays followed by removing unhybridized cDNA
- Removing the unhybridized cDNA
- Detecting and quantitating hybridized cDNA
- Comparing the quantitative data from the various samples
mRNA Isolation
Isolating quality RNA is a critical first step
in successful array analysis. Most array analysis protocols recommend
using mRNA to ensure maximum sensitivity and minimum background signal.
Ambion's Poly(A)Purist™ mRNA
Isolation Kits are ideally suited for this application. These
kits feature an optimized oligo(dT) hybridization that minimizes
rRNA contamination, resulting in high yield and high purity mRNA
for cDNA probe synthesis.
cDNA Synthesis
Once RNA samples have been prepared, they are labeled
for subsequent hybridization and detection on the array. The RNA
is converted to labeled cDNA during reverse transcription or can
be labeled during aRNA amplification (see below). Increasing the
efficiency of the reverse transcription reaction reduces the amount
of input RNA required to generate labeled cDNA for hybridization.
Increasing cDNA length enhances hybridization to the array target
as well as increases signal.
Ambion offers two reverse transcription kits
for converting RNA to labeled cDNA. The EndoFree RT™ Kit provides highly efficient,
first-strand cDNA synthesis and extended cDNA products. This kit optimizes sequence-specific priming, resulting in increased
sensitivity in membrane array hybridizations. The Amino
Allyl cDNA Labeling Kit is used for the generation of fluorescently-labeled
cDNAs for glass array analysis.
aRNA Amplification
Gene profiling using glass arrays typically requires
up to 10 µg of total RNA or 2 µg poly(A) RNA for cDNA labeling and
subsequent hybridization. When starting RNA samples are limiting,
they can be reverse transcribed into cDNA and then amplified by transcription,
generating amplified RNA or 'aRNA'. The MessageAmp™ Premier RNA Amplification
Kit is specifically designed for the generation of aRNA from
small amounts of RNA. All Ambion MessageAmp Kits are based on the patented Eberwine
procedure and can amplify RNA as much as 1000X in a single round.
The MessageAmp Premier Kit incorporates extensive improvements over previous kits with a simplified workflow and more efficient reactions. The kit now consists of a set of master mixes rather than individual reagents, reducing the number of components, pipetting steps, and manipulations.
The MEGAscript™ Kit is
also available for in vitro transcription of cDNA templates to produce
aRNA.
Hybridization and Washing
Hybridization and wash consistency are critical
when comparing signals from different samples. The choice of hybridization
buffers and incubation temperatures can dramatically affect the overall
sensitivity and specificity of the experiment. Ambion has found that
many published array hybridization procedures either lack the sensitivity
necessary to detect rare messages or generate substantial cross-hybridization
on the array. The SlideHyb™ Survey
Kit and Glass Microarray Hybridization Buffers were designed
to enhance glass microarray hybridization while minimizing cross-hybridization.
Detection and Quantitation
Optimizing every step in the array analysis procedure
provides more consistent, reliable, and sensitive data. Once the
array analysis protocol has been completed, the array is analyzed,
via phosphoimaging for membrane arrays, and fluorescent quantitation
for glass microarrays.
Array Controls
In order to compare array data within and between
experiments, it is important to use the proper controls. The ideal
control for array analysis is a sequence that has stable expression,
i.e. does not vary from sample-to-sample. Unlike housekeeping genes
(e.g. ă-actin and GAPDH), the best array controls are unrelated,
exogenous sequences that are found both on the array and in the sample.
A specific DNA sequence is used during the printing of the array
and a complementary RNA transcript is added to the RNA sample to
serve as a control for reverse transcription, hybridization, and
washing. The ArrayControl™ is
a set of products including DNA Oligo Spots (sense and antisense),
PCR Spots and complementary RNA Spikes to control for every step
of array analysis.
Validation
After differentially expressed genes are identified
by array analysis, their differential expression should be confirmed
and quantitated. Ambion offers a wide variety of assays for quantitating
mRNA expression including products for RT-PCR, Northern
blotting, and ribonuclease
protection assays. Explanations of these techniques along with
guidelines for setting up quantitative analysis are available in
the Basics section
of the web site.
Related Articles
| Cat# |
Product Name |
Size |
| AM1330 |
MEGAscript® SP6 Kit |
40 rxns |
| AM1334 |
MEGAscript® T7 Kit |
40 rxns |
| AM1338 |
MEGAscript® T3 Kit |
40 rxns |
| AM1705 |
Amino Allyl cDNA Labeling Kit |
15 rxns |
| AM1710 |
RETROscript® Kit |
40 rxns |
| AM1780 |
ArrayControl™ RNA Spikes |
8 x 1 µg |
| AM1792 |
MessageAmp™ Premier RNA Amplification Kit |
30 rxns |
| AM1908 |
MEGAclear™ Kit |
20 rxns |
| AM1916 |
Poly(A)Purist™ Kit |
6 purifications |
| AM1919 |
MicroPoly(A)Purist™ Kit |
20 purifications |
| AM1922 |
Poly(A)Purist™ MAG Kit |
Isolation from up to 8 mg total RNA |
| AM8861 |
SlideHyb™ Glass Array Hybridization Buffer #1 |
5 x 2 ml |
| AM8862 |
SlideHyb™ Glass Array Hybridization Buffer #2 |
5 x 2 ml |
| AM8863 |
SlideHyb™ Glass Array Hybridization Buffer #3 |
5 x 2 ml |
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