YILIMART - μMACS One-step T7 Template Starting Kit

The µMACS One-step T7 Template Kit facilitates the in-column synthesis of double-stranded cDNA, containing a T7 promotor, from limited biological sample, including biopsies or low numbers of cells. A subsequent in vitro transcription (IVT) can be performed in the same column, thus minimizing loss of sample and concurrently allowing for an efficient amplification of antisense-RNA (aRNA) for microarray gene expression analysis.

Detailed procedure

After cell lysis and clearing of the cell lysate, the mRNA is magnetically labeled with µMACS MicroBeads, containing an attached Oligo(dT)-T7 nucleotide sequence. Due to the small size of the MicroBeads, the hybridization to the poly(A)

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tail of the mRNA molecules is completed within seconds. The sample is loaded onto a µ Column placed in the magnetic field of the thermoMACS Separator. After washing, the magnetically labeled mRNA is retained on the column. A ready-to-use reverse transcriptase enzyme mix is added onto the column and the thermoMACS Separator is set to 42 °C, enabling efficient first-strand cDNA synthesis. Subsequently, a second strand synthesis mix containing DNA polymerase, DNA ligase, and RNase H generates double-stranded cDNA. After thorough washing, pure double-stranded cDNA is eluted from the column.

Optionally, before elution of the cDNA, the antisense RNA can be synthesized on the same column within three hours by adding a T7 RNA polymerase (enzyme is not included in the kit). In this case, the amplified antisense RNA is eluted, purified, and ready for further applications, such as labeling and microarray hybridization.

Figure 1
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Figure 1

Principle of MACS Technology for one-step mRNA isolation, cDNA synthesis, antisense-RNA synthesis, and purification using the µMACS One-step T7 Template Kit.

Principle of MACS Technology for one-step mRNA isolation, cDNA synthesis, antisense-RNA synthesis, and purification using the µMACS One-step T7 Template Kit.
Figure 2
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Figure 2

Amplification of two identical mouse liver samples. The mRNA of two identical mouse liver tissues was isolated and cDNA was generated using 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 Microarrays, mouse, antisense (1076 genes). The resulting regression coefficient was 0.99.

Amplification of two identical mouse liver samples. The mRNA of two identical mouse liver tissues was isolated and cDNA was generated using 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 Microarrays, mouse, antisense (1076 genes). The resulting regression coefficient was 0.99.

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mRNA Isolation Kits average RNA yields

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μMACS One-step T7 Template Starting Kit

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Detailed procedure

Figure 1

Figure 1

Figure 2

Figure 2

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