What are the advantages of the µMACS™ One-step cDNA Kit compared to traditional cDNA synthesis methods?
As the name implies, pure cDNA is generated directly from cells, tissue, or blood in only a single step: The MACS-One-step-cDNA-Labeling-Kits efficiently combines the direct magnetic isolation of mRNA with a revolutionary in-column cDNA synthesis.
In a procedure that takes 90 minutes in total, the poly(A)? tail of eukaryotic mRNA is effectively labeled with paramagnetic, extremely small Oligo(dT) MicroBeads (diameter 50 nm), and then applied to a MACS Column. The column is exposed to the magnetic field of a heatable thermoMACS™ Separator; thus, magnetically labeled mRNA remains in the column, while all other material is washed away. Subsequently, the provided enzyme mix is applied for an in-column reverse transcription. After the enzyme-mix components are washed away, the high-quality cDNA is eluted.
This one-step, in-column procedure minimizes loss of sample, saves time, and is very convenient. The µMACS One-step cDNA Kit can be used for small samples containing only a few cells to as many as 107 cells, 30 mg animal or 100 mg plant tissue, or 200 µg total RNA. The kit contains all necessary buffers, Oligo(dT) MicroBeads, µ Columns, and the ready-to-use reverse transcriptase enzyme mix.
For synthesis of labeled first-strand cDNA the µMACS™ One-step cDNA Labeling Kit is available. Furthermore, a kit for in-column T7 amplification is also available (µMACS One-step T7 Template Kit).
Can further enzymatic reactions be performed in the µ Column, such as cDNA labeling?
With MACS® Technology, many kinds of enzymatic reactions can be performed directly in the column, allowing a fast workflow and minimized loss of sample, as no tube-to-tube transfer of the sample is necessary. Furthermore, the sample purification after the reaction is easily performed without additional phenol extraction or precipitation.
DNase I treatment
When processing cell lysate to mRNA and first-strand cDNA, an in-column genomic DNA removal with DNAse I can optionally be performed. As the µMACS™ mRNA isolation yields mRNA virtually free of genomic DNA contamination, this step is only recommended for very limited samples in combination with highly sensitive downstream applications such as real-time PCR from mRNA isolated from a few cells only, when intron-spanning primers are not available.
RNase H treatment
Although residual mRNA does generally not interfere with downstream PCR or real-time PCR, RNAse H removal of mRNA in mRNA-cDNA hybrids can be performed directly in the column. Thereby, first-strand cDNA without any residual mRNA is eluted.
Labeling of cDNA
The synthesis of cDNA as well as cDNA labeling reactions can be performed directly in the µ Column that is placed in the thermoMACS™ Separator.
The synthesis of ds T7-tagged cDNA and linear T7 amplification can be performed in the µ Column.
How can very low amounts of mRNA be quantified?
1. Quantification by measuring UV absorbance
mRNA yield can be determined by measuring the absorbance (A) at
260 nm, if RNA concentrations >5 ng/µL are expected. The measured
A260 should have a value of =0.1 to ensure reliable analysis. For accurate results with conventional spectrophotometers it is recommendable to use RNase-free disposable cuvettes with a small volume (50 µL), which allow measurement of the undiluted mRNA eluate. An absorbance of 0.1 corresponds to 4 µg RNA/mL (path length: 1 cm). Therefore, the yield of mRNA can be calculated as follows:
A260 × 40 × dilution factor = µg mRNA/mL
For UV measurements of volumes of 1 µL, instruments of NanoDrop Technologies such as NanoDrop ND-100, can be used.
2. Quantification using fluorescent dye
If RNA concentrations <5 ng/µL are expected, measure dilutions of the mRNA eluates with the RiboGreen® RNA quantitation assay (high range from 20 to 1000 pg/µL; low range from 1 to 50 pg/µL, Molecular Probes), in a fluorescence microplate reader (excitation at 500 nm, emission at
525 nm). Please follow the instructions of the manufacturer.
3. Capillary electrophoresis
Using capillary electrophoresis, very low concentrations of RNA can be detected. Detection limits of the products range between 200–5,000 pg/µL RNA (Experion RNA HighSens Analysis Kit, BioRad Laboratories; RNA 6000 Pico LabChip® Kit, Agilent Technologies) and 25–500 ng/µL RNA (Experion RNA StdSens Analysis Kit, BioRad Laboratories; RNA 6000 Nano LabChip Kit, Agilent Technologies).
How much mRNA can be isolated with the µMACS™ mRNA Isolation Kits?
Important aspects concerning mRNA yield from biological samples are cell or tissue type, origin and status, and the intactness of the sample. Hence, immediate disruption of tissue or cells in lysis buffer, quick-freezing, or submersion of cells or tissue pieces in an RNA-stabilizer like PrepProtect™ Stabilization Buffer* is essential. Information about average RNA yields can be downloaded from www.miltenyibiotec.com.
Depending on the source and quantity of the mRNA, there are three different µMACS™ mRNA Isolation Kits to choose from.
Using the MACS® Technology, mRNA can be isolated with very high efficiency. This is particularly important when working with low amounts of starting material, such as rare cells or samples from microdissection, or when rare transcripts have to be isolated.
The MicroBeads effectively label the mRNA molecules in a cell. Even mRNA from five Jurkat cells was successfully isolated using the µMACS mRNA Isolation Kit.
* Submersion of tissue or cells in PrepProtect™ Stabilization Buffer protects RNA and denatured protein from degradation.
Does total RNA have to be isolated beforehand?
No. The µMACS™ Technology allows high-quality mRNA isolation directly from various biological materials. The two main reasons are: i) The extremely small (diameter 50 nm), paramagnetic MicroBeads enable an efficient labeling of mRNA within seconds; while, ii) MACS® Column Technology simplifies the required washing steps. If a two-step mRNA isolation from total RNA is favored, µMACS mRNA isolation is the method of choice to obtain high-quality mRNA.
Can µMACS™ mRNA Isolation Kits be used for bacteria?
No. Prokaryotic mRNA (bacterial mRNA) doesn't contain a polyA-tail. Thus, bacterial mRNA will not be labeled with Oligo(dT) MicroBeads which are included in the µMACS mRNA Isolation Kits. However, it is possible to isolate specific bacterial mRNA by using a complementary biotinylated oligonucleotide and µMACS Streptavidin MicroBeads within the µMACS™ Streptavidin Kit.