Prokaryotic Target Labeling Assay

What specifically has changed in the new protocol?

Some of the major differences between the two protocols are highlighted in the following table:

	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.