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Gene Expression Profiling Using Microarray Kits from Roche Applied Science
The use of gene-expression profiling, a genomic application
measuring mRNA transcript levels of many genes in
parallel, has been growing rapidly over the last few years.
Besides the actual microarrays and instrumentation for
hybridization and imaging, target preparation is of major
importance for reliability and sensitivity of expression-profiling
experiments. Several different target preparation
strategies have been recently developed that are mainly
related to the amount of starting material available.
Benefits of the Roche Applied Science Microarray Kits
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High yield and integrity of labeled cRNA
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Representational amplification of small amounts of RNA for gene-expression analysis
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Seamless workflow from cDNA synthesis to cRNA labeling
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Fast and easy procedure
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Incorporation of hapten- or fluorescence-labeled nucleotides
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Only one labeled nucleotide needed
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Compatible with spotted or in situ synthesized microarrays
Especially in medical research applications, sample material is often limited and therefore target amplification is mandatory. Starting from a few micrograms of total RNA, linear amplification of cRNA utilizing T7 RNA polymerase for the preparation of the labeled target, first described by van Gelder and Eberwine [1], has become a standard procedure (Figure 1 a). This protocol consists of a first- and second-strand cDNA synthesis followed by cRNA in vitro transcription using T7 RNA polymerase. For first-strand cDNA synthesis an oligo(dT) primer linked to the T7 promoter sequence is used. In the next step, this T7 linker allows the linear amplification of the template and the simultaneous incorporation of labeled nucleotides with T7 RNA polymerase, generating singlestranded, labeled cRNA. The complete target-preparation workflow consists of multiple enzymatic steps determining the quality and integrity of the targets.
Experimental Validation
To validate the T7 RNA polymerase-based target synthesis procedure, the Roche Applied Science (RAS) Microarray RNA Target Synthesis Kit (T7) and the kit of a major Supplier (A) were tested on three different human acute myeloid leukemia (AML) research samples and one chronic lymphocytic leukemia (CLL) research sample (Figure 1b).
First, 5 g of total RNA was converted into double-stranded (ds) cDNA using the RAS Microarray cDNA Synthesis Kit (eight independent reactions). One group of ds cDNA samples was purified with the RAS Microarray Target Purification Kit, then amplified with T7 RNA polymerase and labeled with Biotin-UTP, using the RAS Microarray RNA Target Synthesis Kit (T7). The resulting labeled cRNA was purified with the RAS Microarray Target Purification Kit. The ds cDNA of the other sample group was purified according to the Supplier As protocol, followed by in vitro transcription and labeling with Biotin- UTP and Biotin-CTP using Supplier As kit. Purification of the labeled cRNA was carried out according to Supplier As recommendations. For all samples, 10 g labeled cRNA was hybridized to HG-U133A GeneChip Arrays. To estimate the performance of the T7 amplification, the yield of biotin-labeled cRNA and the hybridization results obtained from HG-U133A GeneChip Arrays were analyzed (Table 1).
Fast Procedure with High Efficiency
The use of the RAS Microarray Kits demonstrates a more efficient target preparation procedure that results in a greater integrity of labeled target cRNA.
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