Dynabeads™ mRNA DIRECT™ Purification Kit

There are several reasons why DNA contamination may occur:

- Incomplete DNA shearing.
- Incomplete removal of sample lysate after the hybridization step.
- Insufficient washing and/or removal of wash buffers.
- The ratio of sample to beads was too high.

These are some references on cDNA libraries and RT-PCR:

Jakobsen KS, Haugen M, Sæbøe-Larssen S, Hollung K, Espelund M, Hornes E. Direct mRNA isolation using Magnetic Oligo (dT) Beads: A protocol for all types of cell cultures, animal and plant tissues. In Advances in Biomagnetic Separation, Ed Uhlén, M., Hornes, E., Olsvik Ø., Eaton Publishing. 1994:61-72

Raineri I, Moroni C, Senn HP. Improved efficiency for single-sided PCR by creating a reusable pool of first-strand cDNA coupled to a solid phase. Nucleic Acids Research 1991;19:4010

Raineri I, Senn HP. HIV-1 promotor insertion revealed by selective detection of chimeric provirus-host gene transcripts. Nucleic Acids Res. 1992;20:6261-6266

Sharma P, Lönneborg A, Stougaard P. PCR-based construction of subtractive cDNA library using magnetic beads. BioTechniques 1993;15:610-611

Lee Y-H, Vacquier VD. Reusable cDNA libraries coupled to magnetic beads. Anal. Biochem. 1992;206:206-207

Lambert KN, Williamson VM. cDNA library construction from small amounts of RNA using paramagnetic beads and PCR. Nucleic Acids Research 1993;21:775-776

Aasheim H-C, Deggerdal A, Smeland EB, Hornes E. A simple subtraction method for the isolation of cell- specific genes using magnetic monodisperse polymer particles. BioTechniques 1994;16:716-721

Coche T, Dewez M, Beckers M-C. Generation of an unlimited supply of a subtracted probe using magnetic beads and PCR. Nucleic Acid Research 1994;22:1322-1323

Rodriguez IR, Chader GJ. A novel method for the isolation of tissue-specific genes. Nucleic Acids Research 1992;20:3528

Schraml P, Shipman R, Stulz P, Ludwig CU. cDNA subtraction library construction using a magnet-assisted subtraction technique (MAST). Trends in Genetics 1993;3:70-71

Wada H, Asada M, Miyazaki M, Ilda S, Mizutani S. Application of oligo (dT) Dynabeads for the molecular diagnosis of human leukemia. The John Uglestad Conference I: Magnetic separation techniques applied to cellular and molecular biology, 1991

Larsen F, Solheim J, Kristensen T, Kolstø AB, Prydz H. A tight cluster of five unrelated human genes on chromosome 16q22.1. Human Molecular Genetics 1993;2:1589-1595

The purpose of this step (heating at 65 degrees C for 5 min) is to open up secondary structures in the RNA. If you want to use the Oligo(dT)25 on the beads as primers for your cDNA synthesis and generate solid-phase cDNA, you should omit this step. Start with 50 degrees C (otherwise the mRNA will fall off the beads), then proceed to the 65 degrees C step.

It is possible to generate full-length cDNA from mRNA attached to Dynabeads magnetic beads. We recommend a thermostable reverse transcription kit, so that difficult regions with GC-rich secondary structures are accommodated. However, it is not possible to start the reaction by heating the mRNA on the beads because that will elute the mRNA (A:T base pairs are the least thermostable).

We have used ThermoScript reverse transcriptase, inhouse, with Oligo(dT)25 on the beads as primers. The cDNA synthesis was performed according to the manufacturer's instructions. When using a thermostable reverse transcriptase and the Oligo(dT)25 as primer for first-strand cDNA synthesis, an initial step of incubation at 50 degrees C for 5 min is necessary before proceeding at the recommended elevated temperature. This is to start the cDNA synthesis beyond the A:T hybridization point so that the mRNA doesn't fall off the beads. The resulting cDNA is covalently attached to the bead surface, and the beads with the attached cDNA can be used as template in multiple hybridization reactions.

Lithium chloride is included in Washing Buffer A to ensure that the mRNA remains annealed to the Oligo(dT)25 on the beads while everything else is washed away. The major advantage of using LiCl instead of other chloride salts is that LiCl does not efficiently precipitate DNA, proteins, or carbohydrates and therefore reduces the risk of contamination of the final mRNA preparation with DNA and inhibitors of cDNA synthesis, PCR etc.

LiDS is an ionic detergent, similar in function to SDS. LiDS is included in the lysis buffer is to aid in the lysis of the cells and to denature proteins, and in addition it is an effective RNase inhibitor. If you don't have LiDS in the lab, it is also possible to use SDS, but you may wish to add RNAse inhibitor as well.