GeneChip IVT Labeling Kit

The GeneChip® IVT Labeling Kit (in vitro transcription with MEGAscript® Reagents) was designed for in vitro transcription (IVT) amplification and biotin-labeling to prepare targets for GeneChip® brand arrays.

Double-stranded cDNA containing the T7 promoter sequence is used as a template, generated from either One-Cycle or Two-Cycle Target Labeling Assays.

As part of the completely integrated GeneChip reagent product line for expression analysis, the GeneChip IVT Labeling Kit offers:

• Consistent yield and incorporation efficiency
  Utilizes a proprietary synthetic biotinylated nucleotide analog in the IVT reaction with MEGAscript® reagents from Ambion to obtain high cRNA yield and uniform incorporation efficiency.
• Quality tested and standardized system for excellent performance on GeneChip array
  Designed specifically for GeneChip arrays and quality controlled as a complete system with the other accompanying reagents from Affymetrix.
• Streamlined experimental workflow
  Overnight IVT incubation simplifies the workflow of the entire target labeling assay, providing more flexibility when planning lab work.
• Comparable array sensitivity and intra-assay reproducibility as previous generation protocol
  Comparable global array performance including percent Present call allows straightforward transition to this new, more cost-effective product line.
• Convenient packaging
  The larger 30-reaction kit size matches GeneChip® microarray packaging, making it easier for users to track the lot number of all reagents used in each experiment.

Kit Components

GeneChip® IVT Labeling Kit can also be ordered as part of the complete reagent offering.

IVT Labeling Reaction Protocol

Detailed instruction can be found in the Package Insert of the product.

Please note the following:

• The hybridization cocktail preparation has been modified and optimized with the addition of DMSO, at a final concentration of 10%.
• A new fluidics script has been developed specifically for samples prepared with this IVT Labeling Kit and hybridized to 11µm feature-size GeneChip® arrays.

These conditions are different from previous recommendations. Contact Affymetrix Technical Support for any additional questions.

Biotin-labeled cRNA yield

Figure 1

Average yields and standard deviations from independent labeling reactions are shown (n=9 for HeLa and lung samples, n=2 for skeletal muscle, Universal Human Reference RNA, and brain samples), using the IVT Labeling Kit. Consistent high yields are obtained from all tissues.

Biotin-labeled cRNA length

Figure 2

The length of representative cRNA samples from human brain total RNA was examined on Agilent 2100 bioanalyzer using the Agilent RNA 6000 Nano Assay. Comparable cRNA lengths are derived from either the IVT Labeling Kit or NEN biotinylated nucleotides.

GeneChip® array detection sensitivity

Figure 3

Labeled cRNA targets from 5 µg of human skeletal muscle total RNA, using either the IVT Labeling Kit or NEN biotinylated nucelotides, were hybridized to GeneChip® brand arrays. Average percent Present calls from two technical replicates are shown. Comparable or better array sensitivity was detected with the IVT Labeling Kit on both types of arrays.

For research use only. Not for use in diagnostic procedures.