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Affymetrix Preferred Application Provider Status
To satisfy Affymetrix acceptance criteria, two operators on different instrument platforms were required to process 1 and 2 µg aliquots of total RNA islated from HeLa cells. A total of 240 samples were processed using Affymetrix brand cDNA and IVT reagents. Reaction cleanup was done by filtration using Qiagen MinElute* and RNeasy cleanup kits. The quality of the cRNA product was determined by hybridization of a random sample to 60 Human U133 plus 2 Gene Chips. A summary of the data is shown below.
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BioAnalyzer Electropherogram: cRNA size distribution generated during automated processing of 1 µg and 2 µg total RNA samples
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Affymetrix Human U133 plus 2.0 GeneChip cartridge scan of HeLa total RNA
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Log correlation analysis between replicates of HeLa samples at 1 µg target level generated using GCOS 1.2. Green parallel lines indicate two-fold change
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| Parameter |
Acceptance Criteria |
Observed
(% CV) |
Pass/Fail |
| Yield (1 µg target) |
> 25 µg |
32 µg |
Pass |
| Yield (2 µg target) |
> 25 µg |
52 µg |
Pass |
| % Present |
> 53 (< 5%) |
55.3 (2.3%) |
Pass |
| β-Actin 3'/5' ratio |
(< 10%) |
1.5 (8.2%) |
Pass |
| GAPDH 3'/5' ratio |
(< 10%) |
0.99 (2.7%) |
Pass |
| % False Change |
0.75 |
0.28 |
Pass |
| Pearson Correlation |
0.94 - 0.96 |
0.94 |
Pass |
Summary table of results from 60 GeneChips analyzed. The ArrayPlex generated the quantity and quality of target RNA necessary to meet the acceptance criteria for each of the parameters set by Affymetrix.
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ArrayPlex Enables Newly Trained NIDDK Technician to Score at the Top of the 18-lab Peer Proficiency Study
National Institutes of Health (NIH) Instrument Qualification Study
For the NIH qualification study, target prep was done by a newly trained NIDDK (National Institute of Diabetes and Digestive and Kidney Diseases) technician on the ArraPlex using two sets of six rat total RNA samples (isolated from the tissue pools). A total of 12 samples were processed using Affymetrix cDNA and ENZO IVT reagents. Reaction cleanup was done by filtration using Qiagen MinElute and RNeasy cleanup kits. The quality of the cRNA product was monitored by an outside evaluator, Expression Analysis, Inc., who determined the lab performed at the top of the 18-lab peer profeciency study (see below).
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BioAnalyzer Electropherogram: cRNA size distribution generated from 2 µg total RNA rat tissue pools
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Graphical representation of how the NIDDK lab performed in comparison to the 18-lab peer proficiency study reviewed by Expression Analysis, Inc.
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Measurement
(Lab Average) |
Sample Set #1 |
Sample Set #2 |
Peer Average |
| Noise |
2.00 |
2.01 |
2.40 |
| GAPDH 3'/5' ratio |
1.04 |
1.09 |
1.45 |
| β-Actin 3'/5' ratio |
2.02 |
2.07 |
2.94 |
| % Present |
65.4 |
64.6 |
59.5 |
| Signal Present |
992 |
1016 |
1057 |
| False Positives |
1.24% |
1.48% |
2.56% |
Summary table of results from two sets of 6 GeneChips analyzed using target RNA generated on the ArrayPlex
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ArrayPlex Validation Study
The ArrayPlex validation study was initiated to identify sources of variation in target RNA prep by comparison of one operator on the ArrayPlex vs. two skilled laboratory techinicians performing the process manually. The commercially available Human Reference RNA product from Stratagene (San Diego, CA) was used as the source of total RNA. The samples were processed using Invitrogen cDNA and ENZO IVT reagents, both manually and on the automation platform. A total of 12 samples from each picked for comparison by hybridization to 24 Human U133 Plus 2 GeneChips. The results are shown below.
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Automated |
Manual |
Measurement
(Average) |
Plate 8 wells |
Plate 96 wells |
Techinician 1 |
Techinician 2 |
| Background |
44.2 |
54.5 |
43.9 |
44.7 |
| GAPDH 3'/5' ratio |
0.89 |
0.87 |
0.88 |
0.88 |
| β-Actin 3'/5' ratio |
1.01 |
0.99 |
1.21 |
1.13 |
| % Present |
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2 g |
57.8 |
57.1 |
56.4 |
56.4 |
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5 g |
56.8 |
56.2 |
52 |
56.4 |
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7.5 g |
57.7 |
56.3 |
52.9 |
55.7 |
| Signal Present |
790 |
791 |
802 |
800 |
Summary table of averaged results from each set of 6 GeneChips analyzed using target RNA generated from either automated or manual processing methods. Variation in target RNA prep is revealed in the ANOVA (right).
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