Utilize Dynabeads Oligo(dT)25 for mRNA microarrays

Dynabeads Oligo(dT)25 are optimal for mRNA isolation in expression profiling applications.

The hybridisation kinetics between mRNA and the Dynabeads are not only fast and efficient, but also comparable to that of free solution hybridisation. This ensures negligible sample loss.

The many types of arrays/chips commercially available require different quantities of mRNA. With Dynabeads, the possibility of scaling the isolation protocols allows you to isolate mRNA from any quantity of cells. This approach can therefore be used to prepare the amount of mRNA required for any type of microarrays, macroarrays, and chips. A few selected examples are: the isolation of mRNA for GeneChip arrays (Affymetrix Inc.) (1,2), for Gene Expression Microarray™ (Incyte Genomics Inc) (3, 4), and for different types of self-spotted arrays (5-11). Dynabeads can also be applied directly in certain electronic chips or microfluidic devices (12).

Graphs of Dynabeads used to target genes in testicular cancer

Microarray Using mRNA
Microarray Using mRNA Protein

Figure 1. Functional genomics approach using Dynabeads to find target genes in testicular cancer.Left: Microarray using input mRNA, shows either up- (red) or down- (green) regulated genes in tumour relative to normal testis. Right: Two of the genes were also validated as upregulated at their protein level. Courtesy of R. Skotheim and R. A. Lothe, the Norwegian Radium Hospital, Oslo, Norway.

Dynabeads Oligo(dT)25 mRNA isolation beads target and capture mRNA molecules from a wide range of crude sample types and bypass steps to purify total RNA from mRNA. Other technologies designed to purify total RNA yield ~80% ribosomal RNA (rRNA) and force mRNA to compete with rRNA, transfer RNA, micro RNA, and small cytoplasmic RNA for membrane binding.

Advantages of Dynabeads Oligo(dT)25 beads:

  • Fast and gentle procedure yields pure intact mRNA
  • Extremely pure mRNA isolation
  • Sensitive mRNA isolation from ultra-small starting samples

References

  1. Kielman MF. et al. (2002) Apc modulates embryonic stem-cell differentiation by controlling the dosage of b-catenin signaling. Nature Genetics 32(12):594-605.
  2. Zhang Y. et al. (2001) Reproducible and inexpensive probe preparation for oligonucleotide arrays. Nucleic Acids Res. 29(13):e66.
  3. FitzPatrick DR. et al. (2002) Transcriptome analysis of human autosomal trisomy. Hum. Mol. Genet. 11(26):3249-3256.
  4. Wykoff CC. et al. (2000) Identification of novel hypoxia-dependent and independent target genes of the von Hippel-Lindau (VHL) tumor suppressor by mRNA differential expression profiling. Oncogene 19:6297-6305.
  5. Cameron RA. et al.(2000) A sea urchin genome project: Sequence scan, virtual map, and additional resources. PNAS. 97(17):9514-9518.
  6. Fernandes J. et al. (2002) Comparison of RNA expression profiles based on maize expressed sequence tag frequency analysis and micro-array hybridization. Plant Physiol. 128:896-910.
  7. Pancer Z. (2000) Dynamic expression of multiple scavenger receptor cysteine-rich genes in coelomocytes of the purple sea uchin. PNAS 97(24):13156-13161.
  8. Salin H. et al. (2002) A novel sensitive microarray approach for differential screening using probes labeled with two different radioelements. Nucleic Acids Res. 30(4):17.
  9. Skotheim RI. et al. (2002) New insight into testicular germ cell tumorigenesis from gene expression profiling. Cancer Res. 62:2359-2364.
  10. Sørlie T. et al. (2001) Gene expression patterns of breast carcinomas distinguish tumor subclasses with clinical implications. PNAS 98(19):10869-10874.
  11. Frohme M. et al. (2000) Use of representational difference analysis and cDNA arrays for transcriptional profiling of tumor tissue. Ann. N.Y. Acad. Sci. 910:85-104.
  12. Fan ZH. et al. (1999) Dynamic DNA hybridization on a chip using paramagnetic beads. Anal. Chem. 71(21):4851-4859.
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