The Whole Transcript (WT) Sense Target Labeling Assay Has an Updated Protocol

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Effective July 2007, please promptly replace all WT Sense Target Labeling Assay manuals with the 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
Figure 1
Average Total cRNA Yields using WT version 3 and WT version 4 Protocols

Increase your cRNA yields today

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.

Additional changes to the new WT Sense Target Labeling Assay include:

  • 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.

Scatter Plot of Average Probe Set Intensity (3 reps) WT version 3 vs WT version 4 Protocol
Figure 2
Scatter Plot of Average Probe Set Intensity (3 reps) WT version 3 vs WT version 4 Protocol

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.

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