| Previous Protocol | New Protocol | Reason for Change | |
|
Poly-A RNA internal positive controls |
Detailed protocols are provided for users to prepare their own poly-A transcripts. |
GeneChip Poly-A RNA Control Kit (P/N 900433) is used and instructions for dilution and addition to total RNA samples are included. Note that different dilution procedures are necessary when preparing targets for either the 49-format (such as the GeneChip® E.coli Antisense Genome Array) or the 100-format (such as the GeneChip® P.aeruginosa Genome Array) arrays. Follow the instructions in the Expression Analysis Technical Manual closely for best results. |
More convenient and consistent using a commercial kit which is quality-tested by Affymetrix. |
| cDNA Purification | QIAquick PCR Purification Kit from QIAGEN. | MinElute PCR Purification Kit from QIAGEN. | Reduced elution volume from the previous 40 µL to the current 12 µL for better assay flow to the next cDNA Fragmentation step. |
| cDNA Fragmentation | Total reaction volume of 50 µL. | Reduced total reaction volume to 20 µL | Better assay flow to the next Terminal Labeling step. |
| Terminal Labeling |
Used the Enzo® BioArray™ Terminal Labeling Kit. Different reaction volumes for the different array formats. |
Uses a combination of Promega Terminal Transferase
and the GeneChip DNA Labeling Reagent from Affymetrix. One recommendation for all array formats. |
Consistent supply and consistent quality. Simpler recommendation using one formula for all
array types. |