Novel Priming Method Eliminates Need for rRNA Depletion
The WT Expression Kit is the latest RNA amplification innovation from Ambion®. It enables faster, more sensitive, and highly reproducible results with Affymetrix® whole transcriptome microarrays. The kit is designed to generate amplified sense-strand cDNA ready for fragmentation and labeling using the Affymetrix® GeneChip® WT Terminal Labeling Kit. The Ambion® WT Expression Kit has been optimized specifically for use with Affymetrix® GeneChip® Human, Mouse, and Rat 1.0 ST Arrays, where “ST" stands for sense target, and the probes on the arrays are distributed throughout the entire length of each transcript.
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 employs a novel, patent pending, reverse transcription (RT) priming method that eliminates the need for a separate rRNA 查看更多depletion step. The kit includes RT primers designed with a proprietary oligonucleotide matching algorithm that eliminates primer sequences with homology to known ribosomal RNA sequences. The result is complete and unbiased coverage of the transcriptome, with a significant reduction in rRNA amplification, compared to other methodologies.
Consistent Results With 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—an amount that may be difficult or impossible to obtain from limited samples such as stem cells or small tissue samples, or may require significant time and expense spent culturing cells. The ability to use smaller amounts of RNA enables analysis of precious samples and provides more efficient and cost-effective experimentation for most sample types.
To demonstrate the performance of the Ambion® 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 Ambion® 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 Ambion® 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 is remarkably 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 is also high for both kits (see Product Bulletin for data). This high correlation with real-time PCR results indicates that relative accuracy, as measured with an alternative gene expression platform, is very high for this kit.
Sensitive Expression Profiling
In addition to the lower input RNA requirement and high concordance between the Affymetrix® method and TaqMan® real-time PCR data, the Ambion® 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 Ambion® 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 Ambion® WT Expression Kit. Specifically, 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.
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