Preparation of label from small cell numbers for microarray screening.
Common methods for amplification of labelled cRNA for hybridisation to Affymetrix GeneChips (Affymetrix Inc., Santa Clara, CA) assume that starting material is not limiting and require 2-5 microg of total RNA. However, often the target population of cells under study is a rare subset like stem cells or dendritic cells. To bypass this difficulty in the past, either the whole tissue or a representative cell line was used to obtain enough cells for experimentation. There are obvious limitations with these approaches. In the case of whole tissue, there are contaminating cells types, and cell lines may not exactly reflect cells in vivo. It has been reported that two cycles of amplification can generate enough labelled cRNA for hybridisation from as little as 2 ng of total RNA. This allows Affymetrix technology to be used to screen the gene expression of cells in low number, rare cell subsets or small patient biopsies. Adoption of this approach can be used to give an accurate profile of genes expressed in the specific cell subset of interest. Published methods and successful variations applied to these are discussed here.
Wilson
Heather L
HL
School of Biochemistry and Molecular Biology, Faculty of Science, Australian National University, Canberra, Australia.
O'Neill
Helen C
HC
D009693:Nucleic Acid Hybridization; D020411:Oligonucleotide Array Sequence Analysis; D018075:RNA, Complementary / Q000235:genetics
D018075:RNA, Complementary