Dynabeads™ Oligo(dT)₂₅

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.

Ribosomal RNA is effectively eliminated by reextracting the mRNA from the eluate. Reuse the same Dynabeads Oligo(dT)25 beads that were used for the original isolation. Wash the beads twice in Washing Buffer B. Dilute the eluted mRNA with 4 times its volume of Lysis/Binding Buffer, then add the beads. Incubate with mixing at room temperature for 3-5 minutes, then continue with the Direct mRNA Isolation Protocol.

We recommend immediate use of Dynabeads magnetic beads-mRNA complex or eluted mRNA for cDNA synthesis, in RT-PCR, or for other downstream applications. If storage is needed, we recommend you elute the mRNA from the beads using 10 mM Tris-HCl buffer (pH 7.5) and freeze it(-80°C). It is very important that all equipment and samples are RNase free.

Dynabeads magnetic beads are compatible with TaqMan real-time PCR chemistry and non-capillary real-time PRC instruments. However, Dynabeads magnetic beads exhibit a low level of autofluorescence that can increase the intensity of the fluorescent signal to some degree. This can be compensated for by using a Dynabeads magnetic beads and water background in the instrument. Then background signal intensity can be subtracted from the sample signal intensity in all subsequent real-time PCR experiments containing Dynabeads magnetic beads. Alternatively, when using the standard curve method of analysis, an appropriate amount of Dynabeads magnetic beads can be added to each sample used to construct the standard curve.

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