Ambion® WT Expression Kit, high throughput

Catalog number: 4440537

Ambion™  Related applications: Microarray Analysis

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Description

The Ambion® WT Expression Kit is an RNA amplification kit that is designed to generate amplified sense-strand cDNA ready for fragmentation and labeling using the Affymetrix® GeneChip® WT Terminal Labeling Kit.

Features of the WT Expression Kit:

• Optimized for use with Affymetrix® GeneChip® Human, Mouse, and Rat 1.0 ST Arrays
• Allows RNA samples as small as 50 ng to be analyzed on Affymetrix® GeneChip® Arrays
• Supports a streamlined workflow that does not require a separate rRNA depletion step
• Is ideal for high-sensitivity expression profiling

Eliminates separate rRNA depletion step, preserves transcriptome coverage

The first-generation Affymetrix® amplification method requires depletion of ribosomal RNA (rRNA) from RNA samples for optimal exon-level analysis. Conversely, the Ambion® WT Expression Kit uses a novel reverse transcription (RT) priming method that eliminates the need for a separate rRNA depletion step. The kit includes RT primers designed using a proprietary oligonucleotide matching algorithm that eliminates primer sequences with homology to known ribosomal RNAs. The result is complete and unbiased coverage of the transcriptome, with rRNA amplification levels significantly lower than those of other methods.

Consistent results from less input RNA
With the Ambion® WT Expression Kit, samples as small as 50 ng of total RNA can be analyzed on Affymetrix® GeneChip® Human, Mouse, and Rat Exon and Gene 1.0 ST Arrays. Previous methods required 1 µg of total RNA—often difficult or impossible to obtain from limited sources such as stem cells or small tissue samples. The modest requirement for input RNA permits the analysis of rare samples and provides for more efficient and cost-effective experimentation for most sample types. To demonstrate the performance of the WT Expression Kit, RNA from three sample types (HeLa cells, and Microarray Quality Control (MAQC) A and B samples) was prepared in triplicate using either the Affymetrix® GeneChip® WT cDNA Synthesis and Amplification Kit or the WT Expression Kit, and analyzed on Human Exon 1.0 ST Arrays. Total RNA (1 µg) prepared by the Affymetrix® protocol underwent an rRNA-depletion step while just 50 ng total RNA (20-fold less) was prepared for microarray analysis using the WT Expression Kit. Both sets of samples were handled according to the manufacturers' recommendations. Direct correlation of log2 ratios for MAQC samples A and B was high, (r >0.94) at both the exon and transcript levels. Correlation of log2 ratios with gene expression data obtained using TaqMan® Gene Expression assays for real-time PCR was also high for both kits (see Product Bulletin for data).

Identify more differential expression with less RNA
In addition to its lower input RNA requirement, the WT Expression Kit provides a significant increase in sensitivity. A greater number of probe sets detected above background was obtained at the exon level with the WT Expression Kit as a result of an increased signal-to-noise ratio. A comparison of differential expression analysis results from the two methods indicated that, while differentially expressed genes and exons of MAQC A and MAQC B samples were similar between the two kits (78% and 89%, respectively), significantly more differential expression was observed on arrays hybridized with samples prepared using the WT Expression Kit. 499 more genes and 6,850 more exons were found to be differentially expressed in samples prepared using the Ambion® kit, compared to samples prepared with the Affymetrix® kit.

Note: For high-throughput robotics.
For Research Use Only. Not for use in diagnostic procedures.

Specifications

Includes Label or Dye: No
Labeling Method: Indirect Labeling
Product Size: 24 reactions
Reverse Transcriptase: Proprietary
Sample Type (Specific): Total RNA
Starting Material (Cell #): ≥ 50 ng total RNA
System: Affymetrix GeneChip®

Contents & storage

• 50 µl First-Strand Enzyme Mix (-20°C)
• 160 µl First-Strand Buffer Mix (-20°C)
• 175 µl Second-Strand Enzyme Mix (-20°C)
• 400 µl Second-Strand Buffer Mix (-20°C)
• 210 µl IVT Enzyme Mix (-20°C)
• 750 µl IVT Buffer Mix (-20°C)
• 10 µl Control RNA 1 mg/ml (-20°C)
• 425 µl 2nd-Cycle Buffer Mix (-20°C)
• 100 µl Random Primers (-20°C)
• 275 µl 2nd-Cycle Enzyme Mix (-20°C)
• 100 µl RNase H (-20°C)
• 3.5 ml Nucleic Acid Binding Buffer Concentrate (room temperature)
• 700 µl Nucleic Acid Binding Beads (4°C)
• 15 ml Nucleic Acid Wash Solution Concentrate (room temperature)
• 6 ml Elution Solution (4°C or room temperature)

Documents

Manuals & protocols