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| Protocol Name | Protocol for Affymetrix's GeneChip System: Sample Preparation (nanogram assay) |
| Protocol Type | labeling |
| Home Page | N/A |
| Description |
Protocol was presented at November, 2001 Director's Challenge meeting. It permits
amplification of nanogram amounts of total RNA for hybridization to Affymetrix GeneChips. It is based on a modification of the Phillips
and Eberwine method and performs 2 rounds of in vitro transcription, in which first round cRNA
is used to generate a second round cDNA. Typical yield from 25-50 ng of RNA input is 85 ug of
cRNA. Final round cRNA (15 ug) is fragmented, hybridized, washed, stained, etc. to a GeneChip according to the standard Affymetrix protocol.
Data analysis indicates that: 1. the procedure can be used for amounts down to 25 ng total
RNA; 2. assay noise is 1 to 2-fold higher than the standard amplification procedure; and 3.
amplification of amounts > 100 ng may fail due to insufficient substrates. Total RNA for
the reaction is measured by the Molecular Probes Ribogreen kit.
(Note: protocol is in Excel format.) |
The protocol may be downloaded here.
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