Affymetrix - Product Update, WT Sense Target Labeling Assay

The Whole Transcript (WT) Sense Target Labeling Assay Has a New Version

2007年7月から全てのWT Sense Target Labeling Assayのプロトコルが変わりました。試薬自体に変更はありません。 updated manual (revision four).

The WT Sense Target Labeling Assay is used for both GeneChip® Exon 1.0 ST Arrays and GeneChip® Gene 1.0 ST Arrays. Please use the new manual for both of these products.

Average Total cRNA Yields using WT version 3 and WT version 4 Protocols

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1.cRNAの収量が改善されます

The new updated manual will enhance assay performance through a key change that increases cRNA yield with minimal impact on assay experiment time.

Change: Elute two times through the IVT cRNA Cleanup Spin Column during cleanup after first-cycle, cRNA synthesis.

2.このプロトコル変更で新たに加わった改良点

Additional safe stopping points (optional):
- After RNA Cleanup/Concentration following the RiboMinus rRNA reduction step, before proceeding to first-cycle, first-strand cDNA synthesis [store rRNA-reduced total RNA at -80°C]
- After IVT reaction and the cRNA cleanup step in the first cycle, before proceeding to the second cycle of reverse transcription [store cRNA at -80°C]
- After reverse transcription and the single-stranded cDNA cleanup step in the second cycle, before fragmentation and labeling [store single-stranded cDNA at -20°C]
- After fragmentation and labeling, before hybridization [store labeled cDNA at -20°C]

A broadened range of starting RNA input amounts:

	1µg Total RNA Labeling Protocol	100 ng Total RNA Labeling Protocol
Recommend Amount of Starting Material	1 µg Total RNA	100 ng Total RNA
Acceptable Range of Input Amount	1-2 µg*	100-300 ng
Procedural Difference	Requires rRNA Reduction with RiboMinus Kit	Omits rRNA Reduction
Exon ST Arrays	Recommended	Not Optimal
Gene ST Arrays	Acceptable	Recommended

* When using 2 µg of starting material, it is highly recommended to scale up the RiboMinus reagents to ensure efficient rRNA reduction. Failure to do so may have a small negative impact on sensitivity, particularly at the exon level.

The 1 µg Total RNA Labeling Protocol has been optimized for the 1 µg input amount. However, a modest increase in cRNA yield has been observed by increasing the amount of total RNA used. Input amounts up to 2 µg show no adverse impact on array performance. Note, however, that the protocol still recommends using 1µg of total RNA input. The amount should only be increased if sufficient cRNA yields are not obtained using the recommended 1µg starting amount.

When omitting the RiboMinus procedure, using 100 ng of total RNA as input has been shown to generate sufficient cRNA from a diverse set of RNA sources. However, for some RNAs that yield less cRNA, increasing the amount of total RNA used can result in a modest increase in cRNA made. Target generated from a range of 100-300 ng of input total RNA has demonstrated equivalent array performance.

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Switch now?don't wait!

The new WT Sense Target Labeling Assay can be safely adopted at any point in the experimental process. Switching protocols mid-experiment will have no significant impact on array performance or on gene expression profiles. Please switch your protocol now to version four to achieve higher cRNA yields.

Interested in learning more?
The Affymetrix Product Management and Reagents Development team will be hosting a webinar to introduce the changes behind the new WT Sense Target Labeling Assay. Data supporting the benefits of the modifications will be presented. The session will be interactive, giving you an opportunity to ask any questions about the updated WT assay. Please join us as Affymetrix presents an exciting advance in the quality and power of our WT system.

For more information on the webinar, please register here.