One step only yields pure cDNA directly from cells, tissue, or blood when mRNA is isolated and in-column reverse transcribed with MACS® Technology.
Sensitive analyses even from only a few cells are improved with the fast μMACS™ Procedure that minimizes loss of material.
Three kits in one enable magnetic mRNA isolation, cDNA synthesis, and purification from up to 107 cells.
Ready-to-use reagents and MACS Column Technology make cDNA synthesis especially convenient. |
Pure cDNA in one step. The isolation of RNA with subsequent first-strand cDNA synthesis has never been so easy: The μMACS™ One-step cDNA Kit combines the efficient direct, magnetic isolation of mRNA with a revolutionary in-column cDNA synthesis.
In a procedure that takes 90 minutes in total, mRNA is magnetically labeled and bound to a column, where it is then reverse transcribed and purified, before the high-quality cDNA is eluted.
90 minutes from cell to cDNA. The process, shown in figure 1, takes advantage of two aspects of MACS® Technology: In an instant, the extremely small Oligo(dT) MicroBeads (Ø 50 nm) efficiently label the poly(A)+ tail of eukaryotic mRNA; no hybridization time is required. µ Columns then enable effective washing and processing of magnetically bound molecules.
Taken together, this allows the purification and reverse transcription (RT) of mRNA directly from cells, tissue, or blood. Along the way, the procedure runs with the familiar convenient handling of MACS Technology: Following a single centrifugation to clear the lysate, only a few pipetting steps are required as the reverse transcription reaction is already set up with a ready-to-use enzyme mix.
Optimized sensitivity for any application. As only 1–5% of total RNA are messenger RNA, analysis based on mRNA often give significantly improved results when compared to total RNA. Isolated mRNA shows stronger bands in Northern blots and gives clearer results in microarray hybridizations or quantitative PCR.
In particular, small samples such as biopsies or rare cells, and defined samples, like isolated cell subsets, make accurate and sensitive analyses more and more important. Therefore, µMACS One-step cDNA Kit can be used equally well either for very few cells or for up to 107 cells, 30 mg animal tissue, 100 mg plant tissue, or 200 μg total RNA.
High-quality cDNA. Quantity, purity, and full-length reverse transcription define the quality of cDNA for many applications. MACS Column Technology and the fast one-step procedure minimize the risk of mRNA contamination and loss of material.
The temperature-controlled thermoMACS™ Separator provides the strong magnetic field and quickly adjusts the accurate RT incubation temperature in the µ Column. Further enzymatic reactions like DNase digestion of genomic DNA traces or RNase H degradation of RNA can easily be performed in the column.
Finally, the column-bound first-strand cDNA is purified before elution, avoiding downstream purification procedures and potential loss of cDNA. The high quality of the resulting cDNA is clearly visible in downstream applications. For microarray analysis the μMACS One-step cDNA Labeling Kit enables direct synthesis of fluorescent or radioactive cDNA. |
| |
|
| Figure 2 |
| mRNA isolation and cDNA synthesis from 500 and 5 Jurkat cells (A). Analysis by PCR amplification of GAPDH fragment (B). |
| A: mRNA was isolated and reverse transcribed into cDNA by μMACS One-step cDNA Kit (a: 500 cells; b: 5 cells) or by conventional tube methods (c: 500 cells; d: 5 cells). e: non-template control. 10% of each cDNA was analyzed by quantitative PCR using intron-spanning primers. |
 |
| B: μMACS One-step cDNA synthesis (lane 1 and 2: 500 Jurkat cells; lane 3 and 4: 5 Jurkat cells, M: 100 bp ladder). 50% of each cDNA was used in a standard PCR reaction. A 372 bp fragment of GAPDH was amplified with intron-spanning GAPDH primers (genomic DNA would give a 476 bp fragment) and analyzed by agarose gel electrophoresis. |
 |
|
|
