GeneChip® IVT Labeling Kit (in vitro transcription with MEGAscript® Reagents)
The IVT Labeling Kit 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
Component name
Quantity
Volume
10X IVT Labeling Buffer
1
120 µL
IVT Labeling Enzyme Mix
1
120 µL
IVT Labeling NTP Mix
1
360 µL
3'-Labeling Control (0.5 µg/µL)
1
10 µL
RNase-free Water
1
910 µL
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
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
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
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.
