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Efficient and Reliable Linear,,, Amplification of cRNA

lizing 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 accor
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