μMACS™ One-step T7 Template Kit
Linear T7 amplification with paramagnetic beads – The revolutionary process simplifies T7 cDNA template synthesis from limited biological samples like biopsies or low numbers of cells. In-column cDNA template generation and in vitro transcription reduce pipetting steps to minimize loss of sample. Reproducibility for aRNA amplification – The convenient MACS® Technology and ready-to-use reagents reduce operator variance, hands-on time, and template preparation variability. Speed up microarray experiments- μMACS™ One-step T7 Template Kit enables the in-column generation of double strand cDNA with T7 promoter from cell or tissue samples in 140 minutes. A subsequent in vitro transcription (IVT) can be performed in the identical column; thus, minimizing loss of sample and efficiently amplifying aRNA for microarray analysis (please note that IVT components are not included in the μMACS One-step T7 Template Kit). Using MACS Technology, reliable gene expression analysis can be performed from small biological samples down to 5×104 cells or 250 ng total RNA. The typical, up to 1,000-fold amplification yields suffcient aRNA for common microarray analyses: approximately 2–4 μg aRNA from 250 ng total RNA or up to 10–20 μg from 1 μg total RNA. MACS® Technology for T7 template generation. First, extremely small, superparamagnetic MicroBeads with an attached Oligo(dT)-T7 sequence are added to the cell lysate. When this lysate is applied to a μ Column placed in a heatable magnet, the thermoMACS™ Separator, the MicroBead-mRNA complexes are retained in the strong magnetic field. After several washing steps, a ready-to-use reverse transcriptase enzyme mix is added onto the column to synthesize first strand cDNA at 42 °C. Subsequently, a second strand synthesis mix containing DNA polymerase, ligase and RNase H generates double stranded (ds) cDNA. After purification of the ds cDNA, it is possible to perform an IVT reaction with T7 RNA polymerase (enzyme not included in the μMACS One-step T7 Template Kit) on the same column within three hours. In this case, amplified RNA can be eluted, purified, and is ready for further applications like labeling and microarray hybridization, e.g. with PIQOR™ Microarrays, antisense. Efficient in-column synthesis reactions. The fast and convenient μMACS procedure reduces the risk of RNA contamination and degradation. Additionally, in-column reactions minimize loss of sample that generally occurs during tube-to-tube transfer, precipitation, and washing steps – this is especially important when working with minor amounts of sample material. With its highly active reverse transcriptase, the μMACS One-step T7 Template Kit is designed to yield full-length cDNA to be used for in-column aRNA amplification. For a scheme of in-column aRNA amplification with μMACS One-step T7 Template Kit, refer to figure 1. Columns μ Columns (included in kit) Further information Average RNA yields [PDF; 87,7 KB]		Figure 1 Principle of μMACS One-step T7 Template Kit Figure 2 Amplification of two identical mouse liver samples Two identical mouse liver tissues individually amplified with the μMACS One-step T7 Template Kit in combination with the Microarray RNA Target Synthesis Kit (T7), Roche Diagnostic GmbH. Samples were labeled with Cy3 and Cy5 fluorescent dyes, respectively. 500 ng of each labeled aRNA were hybridized to PIQOR Immunology Microarray, mouse, antisense (1,076 genes). The resulting regression coefficient is 0.99. Figure 3 Comparison of traditional versus μMACS T7 RNA amplification T7 amplification products were generated from 1 μg of mouse liver total RNA, reverse transcribed, and analyzed. Electropherogram of amplified RNA from the standard protocol in tubes is shown in purple and from the in-column procedure with μMACS Technology in black (Bioanalyzer 2100, Agilent Technologies Inc).

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