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Keck
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Expression Analysis Experiments
GENECHIP EXPRESSION ANALYSIS EXPERIMENTS
GeneChip Expression analysis experiments
involve the following major steps:
- Experimental Design
- RNA Isolation
- Target cRNA Preparation
- Hybridization to the Test Array
- Test Array Data Analysis
- Hybridization to the Standard
Array
1. Experimental Design:
It is absolutely essential to plan and execute the experiments with utmost care. It is important to begin the planning of the microarray experiment with a proper
question. The experimental model/system should be well-characterized or
well-defined with an independent experimental verification. For example,
if a growth factor was added which induces differentiation in 24-48 hours, but
you are collecting RNA at three hours post-treatment, you should still check a
parallel culture for verification that differentiation occurred at the 24-48
hours period. If possible, a quick check for a gene that is known to be affected
by the treatment should be performed. It is recommended that all
experimental treatments be carried out in triplicates to compensate for
biological and experimental variation. In vitro experiments using
cultured cells should be conducted three different times (not three replicates
performed on the same day) strictly following the same experimental procedures.
Tumor specimens should be devoid of adjacent tissues and if possible, microdissected to obtain as pure a tumor sample as possible. Cell
populations may also be further purified using cell-sorting techniques such as FACS. Dead cells also should be removed by density centrifugation. For
comparative gene expression analysis, it is essential that all the experimental
conditions such as temperature, CO2, media, reagents, and sample processing be
kept identical for all samples.
2. RNA isolation: The
quality of the RNA is the single most important determinant of a successful GeneChip analysis assay. Particularly, differential degradation of RNA can
lead to erroneous conclusions about both the relative and absolute mRNA levels
in the specimens. Although either mRNA or total RNA can be used as starting
material, we prefer total RNA for two reasons: (1) isolating total RNA is easier
and more economical than isolating mRNA and (2) there is loss of starting
material during mRNA purification and consequently more mRNA is required to
achieve sensitivity similar to that of the total RNA. In addition, there
may be differential loss of individual mRNAs.
We recommend TRIzol reagent for isolation of
total RNA from tissue specimens as well as cultured and blood cells. Total RNA
isolated using TRIzol should be further purified using the Qiagen RNeasy cleanup
procedure.
The minimum amount of total RNA required for
GeneChip analysis is 5 µg (When RNA amount is limiting, as little as 2 µg
of total RNA could be used). The A260/A280 ratio
should be at least 1.8 for pure RNA. The quality of RNA should also be assessed
by agarose gel electrophoresis. The Bioanalyzer gel profile should exhibit a
28S band that is 2 times more intense than 18S ribosomal RNA (Figure 2).
It is important that the total RNA is free of
genomic DNA contamination. We have written Standard Operating Procedures for the isolation
of total RNA using both TRIzol and Qiagen RNeasy methods. If genomic DNA contamination is present, it is
essential to remove it by DNase treatment, a modification included in the RNeasy
cleanup protocol.
3. Target (labeled cRNA)
Preparation: High quality total RNA is used as starting material to obtain
labeled cRNA. In the first step, single stranded cDNA is synthesized by
reverse transcription using the poly (A) RNA present in the starting total RNA
sample. Single stranded cDNA is then converted into double stranded cDNA
and purified using the Affymetrix Cleanup Module. An
in vitro transcription (IVT) reaction is then carried out overnight in the presence of biotinylated UTP and CTP to produce biotin-labeled cRNA from the double stranded
cDNA. The resulting cRNA is fragmented in the presence of heat and Mg+2
before hybridization to the test array. Due to the high cost of
cDNA and cRNA synthesis reactions, we use a very strict quality control measures
during the target preparation procedures (Figure 1). At each step the
quality of sample is accessed by Agilent Bioanalyzer (Figure 2).
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