µMACS SuperAmp Kits

µMACS SuperAmp Kits

The µMACS SuperAmp Kit facilitates fast and effective mRNA isolation, cDNA synthesis, and a millionfold amplification of the mRNA-derived cDNA by global PCR starting with 1 to 10,000 cells or appropriate amounts of tissue, without prior preparation of total RNA. Pure mRNA is directly isolated from cells, tissue, or blood by magnetic labeling with µMACS Oligo(dT) MicroBeads. Due to the fast reaction kinetics the small (50 nm) µMACS Oligo(dT) MicroBeads instantly bind to mRNA molecules and allow for efficient and reproducible isolation of high-purity mRNA. Subsequent cDNA synthesis, and cDNA purification take place in the same column, thus reducing loss of material and increasing the sensitivity of analysis. The synthesis of uniform sized cDNA fragments and their 3’ and 5’ tailing allows for a homogenous and uniform amplification of the cDNA during single-primer PCR, thus reducing PCR bias.

Background information

The µMACS SuperAmp Kit enables the generation of double-stranded, labeled cDNA from 1 to 10,000 cells for subsequent whole genome microarray analysis.
Any type of rare cells can be used as starting material:
  • Cell lines
  • Cells sorted with MACS Technology
  • Cells sorted by flow cytometry
  • Tissue biopsies
  • Laser-capture microdissected cells

Detailed procedure

After cell lysis and clearing of the cell lysate, the mRNA is magnetically labeled with µMACS Oligo(dT) MicroBeads. Due to the small size of the MicroBeads, the hybridization to the poly(A) 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. The First-strand cDNA Synthesis Mix is applied to the mRNA and the thermoMACS Separator is set to 42 °C, enabling efficient reverse transcription with random primers that ensure the generation of short, uniform-sized cDNA fragments. After thorough washing, the Tailing Mix is applied to the cDNA. The cDNA is eluted by removing the column from the thermoMACS Separator. After elution, a single-primer PCR amplification, and a Klenow labeling reaction of the cDNA are performed. The reliable SuperAmp Technology is much faster than two rounds of T7 amplification, taking only ten hours from lysed cells to amplified, labeled, and purified DNA.
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