TOTAL RNA PREPARATION
At least ten micrograms of total RNA is isolated from indicated tissues or cell lines using Trizol reagent (Life
Technologies) following the supplier’s protocol. RNA is then treated with RNase-free, DNase (Life Technologies) and further
purified using the RNeasy total RNA clean up protocol (Qiagen). Purified, DNA-free RNA is assessed for integrity by
formaldehyde / agarose gel electrophoresis and quantitated by 260 nm absorbance.
TOTAL RNA REACTION CONTROL
An aliquot of cRNA control mix is added to ten micrograms of purified total RNA. The cRNA control mix is provided
by the core facility and is composed of a set of four, in vitro transcripts generated by T3 RNA polymerase. These
polyadenylated transcripts encode the B. subtilis genes thr, trp, phe, and lys. Probes corresponding to these bacterial
transcripts are represented on all Affymetrix GeneChips (including test chips). Each synthetic transcript is quantitated
and represented at a copy number of 5x106 to 5x108, corresponding roughly to the expected dynamic range of detection
for the GeneChip system. This set of control transcripts allows for the monitoring of cDNA synthesis and in vitro
transcription of biotinylated targets and also provides a reference sample for normalizing between experiments.
cDNA SYNTHESIS
RNA is converted to double stranded cDNA using the SuperScript Choice system (LifeTechnologies) following the
supplier’s protocol except that an HPLC-purified T7-dT24 primer (Genosys) is utilized for first strand synthesis. After
synthesis, the double stranded cDNA is purified by phenol/chloroform extraction and ethanol precipitation.
BIOTINYLATED cRNA SYNTHESIS
The purified cDNA is used to generate the biotinylated cRNA target using the Bioarray High Yield RNA transcript
labeling kit (Enzo) and following the supplier’s protocol. The labeled cRNA is then purified using the RNeasy total RNA
clean up protocol (Qiagen) and quantitated by 260nm absorbance.
FRAGMENTAION
Twenty micrograms of cRNA is fragmented by heating at 94C for 35 minutes in fragmentation buffer (40 mM Trisacetate pH 8.1; 125 mM KOAc; 30 mM MgOAc). An aliquot of fragmented and unfragmented cRNA is analyzed by
formaldehyde/agarose gel electrophoresis to ensure appropriate size distribution (average size: 700 bp unfragmented and
100 bp after fragmentation).
HYBRIDIZATION
Fifteen micrograms of fragmented cRNA is added to a hybridization ‘cocktail’ containing 50 pM of control
oligonucleotide B2 (Genosys), 1X control cRNA mix, 0.1 mg/ml herring sperm DNA, and 1X MES hybridization buffer in a
volume of 300 ul. The control oligonucleotide B2 (5’-biotin:GTCGTCAAGATGCTACCGTTCAGGA-3’) is designed to
hybridize to structural features on the chip to allow for proper scanning and grid alignment. The control cRNA mix is
provided by the core facility and is composed of a second set of four, biotinylated, in vitro antisense transcripts of cDNAs
encoding the E. coli biotin synthesis genes bioB, bioC, bioD, and the P1 bacteriophage cre recombinase gene. Probes
corresponding to these bacterial transcripts are also represented on all Affymetrix GeneChips (including test chips).
Each synthetic transcript is quantitated and represented at a copy number of 2x10 8 to 2x1010, corresponding roughly to
the expected dynamic range of detection for the GeneChip. This set of control cRNAs allows for the monitoring of
hybridization, washing, and staining conditions and also provides a second set of reference samples for normalizing
between experiments. The hybridization cocktail is heated to 95C, 45C, and then one hundred microliters are applied to
a Test3 GeneChip array. The remainder of the cocktail is stored at -20C and the test chip is hybridized at 45C for 16
hours.
WASHING / STAINING/ SCANING
After hybridization, the hybridization cocktail is removed from the chip and stored at -70C. The chip is immediately
placed in the Affymetrix GeneChip Fluidics Station 400 and washed using the preprogrammed steps “mini_euk2”. This
wash program involves a low stringency wash, high stringency wash, steptavidin/phycoerythrin stain, low stringency
wash, anti-streptavidin antibody stain, a second steptavidin/phycoerythrin stain, and a final low stringency wash. After
washing and staining, the test chip is placed in the Affymetrix GeneChip array scanner and image data is captured and
converted to numerical output using the Microarray Analysis Suite version 5.0.
CONTROL PERFORMANCE
For the test chip, the levels of bioB, bioC, bioD, cre, phe, trp, lys, and thr will be assessed. All transcripts should be called
‘present’ and the average difference values of each should be in approximate proportion to their absolute copy number.
Successful signal from bioB, bioC, bioD, and cre, but sub optimal signal from phe, trp, lys, and thr suggests that cDNA synthesis
and in vitro transcription was inefficient. Poor signal from all controls suggests a hybridization, washing, or staining
failure. Normally, the bioB hybridization control (representing 2x108 transcript copies) will be at or slightly above the
level of background noise. The relative expression level of GAPDH and B-actin will also be assessed. The test chip
contains probes directed at the 5’ and 3’ end of these genes for human, mouse, and rat species. In general, the signals from
5’ directed probe sets should be no lower than 50% of those from 3’ directed probe sets. A 3’/5’ ratio of less than 3.0
suggests that cDNA synthesis may have been less than efficient.
EXPRESSION ARRAYS
If the labeled cRNA target passes all metrics of the Test3 chip, it is again heated to 95 °C, 45 °C, and hybridized to the
GeneChip of interest. Hybridization, washing, staining, and scanning are repeated as described above except the
automated fluids program “euk2” is used. The labeled cRNA target may be repeatedly hybridized on 4-6 chips.
Available expression arrays: Human, Mouse, Rat, Drosophila, Arabidopsis, E. Coli, P. aeruginosa
DATA RETREIVAL
Acquired experimental data from the chip is analyzed using the GeneChip Microarray Suite version 5.0. For
comparison between two chips, the ‘baseline chip’ intensity values are measured and normalized to the average signal
intensity. Intensity values of the ‘experimental chip’ are then compared to the baseline chip and a ‘difference change’ is
calculated. Alternatively, hybridization signals may be normalized using the specific expression signals from the set of
bacterial control transcripts. The output of the GeneChip software provides a qualitative call: ‘increase’, ‘marginal
increase’, ‘no change’, ‘decrease’, and ‘marginal decrease’ as well as discrete numerical metrics used to make the call.
Absolute analysis of individual chips is also available. This provides absolute levels of expression for each gene
correlated to a confidence interval. This allows the user to gain a better understanding of how the change results are
generated. Expression elements may be ranked based on absolute level of expression or relative change in expression
between a two chip comparison. All numerical data may be exported to a Microsoft Excel spreadsheet for further
analysis. Expression elements are identified by a GenBank accession number and the GeneChip analysis software allows
for immediate hyperlink to GenBank or the Affymetrix NetAffx website for full sequence annotation.
DATA SUPPORT
The core facility utilizes GeneChip Data Mining Tool version 3.0 and Silicon Genetics GeneSpring version 5.1. These
software packages are available through our local access network during normal business hours. The core also provides
continuous software and data analysis support for both on and off campus users.
GRAPHIC OVERVIEW OF GENECHIP PROCESS
HOW A CHIP REACTS WITH THE SAMPLE
Each of the 16-20 oligonucleotide pairs for any gene on the GeneChip is built by photolithography.
The GeneChip is then ready for the sample.
SAMPLE GRAPHIC OUTPUT
Actual scan of the GeneChip
Intensity values for each probe pair for gene 16637
Correlation clustering graph for a repeated sample
SAMPLE DATA OUTPUT