cRNA target preparation for microarrays: comparison of gene expression profiles generated with different amplification procedures

Microarray technology has become a standard tool for generation of gene expression profiles to explore human disease processes. Being able to start from minute amounts of RNA extends the fields of application to core needle biopsies, laser capture microdissected cells, and flow-sorted cells. Several RNA amplification methods have been developed, but no extensive comparability and concordance studies of gene expression profiles are available. Different amplification methods may produce differences in gene expression patterns. Therefore, we compared profiles processed by a standard microarray protocol with three different types of RNA amplification: (i) two rounds of linear target amplification, (ii) random amplification, and (iii) amplification based on a template switching mechanism. The latter two methods accomplish target amplification in a nonlinear way using PCR technology. Starting from as little as 50 ng of total RNA, the yield of labeled cRNA was sufficient for hybridization to Affymetrix HG-U133A GeneChip array using the respective methods. Replicate experiments were highly reproducible for each method. In comparison with the standard protocol, all three approaches are less sensitive and introduced a minor but clearly detectable bias of the detection call. In conclusion, the three amplification protocols used are applicable for GeneChip analysis of small tissue samples.     

Evaluation of procedures for amplification of small-size samples for hybridization on microarrays

Various approaches have been developed for the preparation of samples for gene expression monitoring. For Affymetrix chips, a standard protocol is widely used; however, this is inefficient for small samples such as laser capture microdissections. Several amplification procedures for such samples already exist, and our goal was to test two of them: the first is based on random PCR amplification, and the second, linear amplification, involves performing the standard protocol twice. We analyzed a dilution of a commercially available mouse brain total RNA preparation and microdissections from mouse hippocampus and striatum. We evaluated the quality of microarray data by analyzing several chip parameters and performing multiple comparisons. At the biological level, brain microdissections prepared with either method gave similar expression results. At the technical level, analysis of the commercial sample showed that random PCR amplification is more reproducible, requires smaller RNA input, and generates cRNA of higher quality than linear amplification.     

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.