mRNA Isolation Kit
For general laboratory use.
The mRNA Isolation Kit isolates highly purified mRNA from cells or tissue.
- Save time. Isolate mRNA directly from cell lysates and tissue homogenates (isolation of total RNA not required).
- Accommodate a wide range of samples. Work with both small and large scale preparations of mRNA.
- Achieve highly effective purification. Generate mRNA that is intact and free of DNA and other RNAs.
- Increase safety. Eliminate the use of hazardous organic solvents.
- Lysis Buffer
- Streptavidin-Coated Magnetic Particles
- Oligo (dT)20 Probe, biotin-labeled
- Washing Buffer
- Water, double-distilled, PCR Grade
- Storage Buffer
The mRNA Isolation Kit prepares highly purified poly (A) + RNA which may be used directly in many molecular biology applications:
- RT-PCR (see Figure 1)
- cDNA synthesis
- Northern blotting
- Northern ELISA
- RNase protection assay
- In vitro translation
Figure 1: Detection of ACE-specific mRNA in chicken eye tissue by RT-PCR. mRNA was isolated from chicken eye tissues with the mRNA Isolation Kit. The mRNA was subjected to RT-PCR, using primers specific for the angiotensin converting enzyme (ACE) gene sequence. Amplification products were analyzed on a 1.5% agarose gel.
Lane 1: Retina
Lane 2: Choroid
Lane 3: Pecten
Lane 4: Iris / ciliary body
Lane 5: Optic nerve
Lane 6: Amplicon after Pvu II digestion
Lane M: DNA Molecular Weight Marker XIV (100 bp ladder)
Lane C: Negative control
Result: A single 525 bp amplicon could be detected in all tissue samples.
Affinity purification is a versatile and highly specific technique for the purification of all classes of biomolecules utilizing differences in biological activities of chemical structures. The high selectivity of this technique results in good purification and high recovery. Often a concentrating effect is reached which enables large volumes to be conveniently processed. The mRNA Isolation Kit depends upon the affinity of the poly(A) + tail of mRNA for a biotin-labeled oligo (dT) probe. The probe can "pull" the mRNA selectively from a lysate without interacting with other RNA or DNA. Once formed, the biotinylated dT-A hybrids can be immobilized on solid surfaces that have been coated with streptavidin, and then washed free of unbound contaminants.
Cultured cells: 2 x 107
Tissue: 200 mg
Total RNA: 500 μg
* Larger or smaller samples may also be used.
Time Required: Approximately 30 minutes
Product: Poly(A)+ RNA, in double-distilled water
Typical Total RNA/mRNA Recovery
|Starting Material and Quantity||Yield/ Recovery Total RNA (µg )||Yield/ Recovery mRNA (µg)||Time Required|
|Cultured cells (107)||30 - 500||0.3 - 25||30 minutes|
|Mouse brain (100 mg)||200||7|
|Mouse liver (100 mg)||700||14|
|Mouse lung (100 mg)||130||10|
The poly (A)+ tail of mRNA hybridizes to a biotin-labeled oligo(dT) probe. Streptavidin-coated magnetic particles capture the biotinylated hybrids and the particles are separated using a magnetic particle separator. Contaminants are removed by washing, followed by elution of the mRNA from the particles using water.
Tested for absence of RNases according to the current quality control procedures; function tested.