Description
Background information
The use of small samples, such as biopsies or rare cells, and defined samples, including isolated cell subsets, makes accurate and sensitive analysis more and more important. Using mRNA instead of total RNA as starting material often yields significantly improved results in Northern blot analysis, microarray hybridization analysis, or quantitative PCR. Regardless of whether samples contain only a few or up to 107 cells, 30 mg animal tissue, 100 mg plant tissue, or 200 µg total RNA – the µMACS and the MultiMACS One-step cDNA Kits are equally well suited for any of these applications.
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 MACS Column placed in the magnetic field of the thermoMACS Separator. During the washing procedure, 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. After thorough washing, pure cDNA is eluted from the column.
The µMACS One-step cDNA Synthesis Kits are designed for manual low-throughput mRNA isolation and cDNA synthesis whereas the MultiMACS One-step cDNA Synthesis Kits are designed for high-throughput applications in a 96-well format. The MultiMACS Kits are used in combination with the MultiMACS M96 Separator that can be integrated in a robotic system to achieve a fully automated approach.
Overview
The µMACS and MultiMACS cDNA Synthesis Kits facilitate fast and efficient mRNA isolation, cDNA Synthesis, and cDNA purification, all in one column, 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. mRNA isolation, cDNA synthesis, and cDNA purification take place in the same column thus reducing loss of material and increasing the sensitivity of analysis. As few as five cells are sufficient to obtain cDNA for PCR analysis.
