Subtractive hybridization is a powerful technique to study gene expression in specific tissues or cell types or at a specific stage. Traditional procedures often are technically demanding and labor-intensive methods that require large amounts of mRNA and might give rise to falsely positive and unreproducible results.

Solid-phase cDNA

Dynabeads Oligo(dT)25 can be used to produce subtracted cDNA probes for screening and isolation of rare, differentially expressed mRNAs. Instead of eluting the captured mRNA off the beads, the bead-bound oligo-dT sequence is used to prime cDNA synthesis to produce a solid-phase cDNA libraries specific for a particular cell type or tissue (1-11).

Advantages

  • A simplified, fast and reliable method for generating subtracted probes or subtracted cDNA libraries
  • Only small amounts of sample/mRNA are required
  • Magnetic handling minimises losses at each step
  • Specific mRNAs are highly enriched
  • Magnetic handling enables simple and rapid buffer changes to optimize conditions for hybridisation and specific enzymatic reactions
  • The subtractor Dynabeads can easily be regenerated, stored and reused

Method I

mRNA from the target material is hybridized with first-strand cDNA from the subtractor material immobilised on Dynabeads. The subtracted mRNA is left in the supernatant after magnetic separation of bead-bound subtractor cDNA with captured common mRNA, and the subtractor beads can be reused. After the final hybridisation step, the subtracted specific mRNA is reverse transcribed to radio-labelled cDNA and used to screen cDNA libraries (5, 10) or for cDNA cloning (9). When the difference between the subtractor and the target mRNA population is small, large amounts of target-specific mRNA may be difficult to obtain and the PCR-based method of Lambert (11) would be more appropriate. See this figure for how it works.

Method II

An alternative approach is to create immobilised cDNA libraries from both target and subtractor mRNA (2, 4). Second-strand cDNA is synthesised by random priming of target cDNA and the fragments eluted and mixed with excess immobilised subtractor cDNA. Common fragments are annealed and removed, while the unique fragments left in the supernatant are used as a probe to screen cDNA libraries (2, 8).If the amount of mRNA is limited or the two mRNA sources are very similar, the material might be insufficient for several rounds of subtraction to be performed, or the material remaining for screening purposes might be insufficient. This can be solved by allowing the subtracted cDNA fragments to reanneal back onto the immobilised subtractor cDNA. The double-stranded cDNA produced is then cut, linkers ligated and the fragments amplified by PCR (3). See this figure for how it works.

References

  1. Camerer E et al. Binding of Factor VIIa to tissue factor on keratinocyte induces gene expression.J. Biol. Chem. 2000;275(9):6580-6585.
  2. Rodriguez IR and Chader GJ. A novel method for the isolation of tissue-specific genes. Nucl.Acids.Res.1992;(13):3528.
  3. CocheT, Dewez M and Beckers M-C. Generation of an unlimited supply of a subtracted probe using magnetic beads and PCR. Nucl. Acids Res. 1994;22(7):1322-1323.
  4. Schoen TJ et al. Isolation of candidate genes for macular degeneration using an improved solid-phase subtractive cloning technique. Biochem.Biophys.Res.Commun.1995; 21(1)181-188.
  5. Aasheim H-C, Logtenberg T and Larsen F. Subtractive hybridization for the isolation of differentially expressed genes using magnetic beads. Meth. Mol. Biol. 1996; 69:115-128.
  6. Leygue ER, Watson PH and Murphy LC. Identification of differentially expressed genes using minute amounts of RNA. BioTechniques 1996;21(6):1008-1012.
  7. Hampson N, Hampson L and Dexter TM. Directional random oligo-nucleotide primed (DROP) global amplification of cDNA: its application to subtractive cDNA cloning. Nucl. Acids Res. 1996;24 (23): 4832-4835.
  8. Schraml P, Shipman R, Stulz P, Ludwig CU. cDNA subtraction library construction using a magnet-assisted subtraction technique (MAST). Trends Genet 1993;9:70-71.
  9. Sharma P, Lönneborg A, Stougaard, P. PCR-based construction of subtractive cDNA library using magnetic beads. BioTechniques 1993;15(4):610-611.
  10. Aasheim H-C, Deggerdal A, Smelang EB, Hornes E. A simple subtraction method for the isolation of cell-specific genes using magnetic monodisperse polymer particles. BioTechniques 1994;16(4):716-721.
  11. Lambert KN and Williamson VM. DNA library construction from small amounts of RNA using paramagnetic beads and PCR. Nucleic Acid Res 1993;21:775-776.