Analysis of gene expression profiles by microarray-based methods in samples with limited cells or tissue, such as those from biopsies, flow cytometry, or laser capture microdissection, often requires linear amplification of the mRNA present in the sample to generate sufficient quantities of labeled product. Studies have shown that linearity can be efficiently maintained through 2 cycles of amplification consisting of cDNA synthesis followed by
in vitro transcription to generate cRNA. The Affymetrix GeneChip® Two-Cycle Target Labeling and Control Reagents provides reagents and purification columns for 2 rounds of linear amplification of 30 samples, starting with purified total cellular RNA. Proprietary agreements also allow Affymetrix to include with the kit, biotin-conjugated nucleotides incorporated in the final
in vitro transcription reaction and required for downstream fluorescence labeling and detection.
Linear amplification and array hybridization can be performed with as little as 10 - 100 ng of total cellular RNA, and requires approximately 3 days to complete. In the first cycle (day 1), a cDNA copy of the mRNA present in the starting sample is synthesized, followed directly by an in vitro transcription (IVT) reaction without the need for a cDNA cleanup step. One drawback lies in the requirement for users to purchase the MEGAscript® T7 Kit (cat# AM1334) directly from Ambion to complete the IVT step in the first cycle. The second cycle (day 2) involves another round of cDNA synthesis, utilizing the cRNA generated in the first cycle as template. The subsequent IVT reaction in the second cycle is performed overnight (16 hr) and incorporates biotin-labeled nucleotides into the final product. The biotin-labeled cRNA is then fragmented in MgCl2 buffer supplied with the kit (day 3) before overnight hybridization to Affymetrix GeneChip® microarrays.
15 ug of the fragmented, biotin-labeled cRNA product are incorporated into a hybridization cocktail that is incubated for 16 hr at 45°C within the encased GeneChip® in a rotating oven. Following hybridization, the arrays are washed, incubated with strepavidin-phycoerythrin conjugate, washed again, and then scanned to generate digital images and raw fluorescence intensity data utilized by computer software programs to quantify gene transcription. The GeneChip® Two-Cycle Target Labeling and Control Reagents also include a poly-A RNA control module and hybridization controls to allow assessment of the amplification and overnight hybridization efficiencies, respectively.
We have utilized the Affymetrix GeneChip® Two-Cycle Target Labeling Kit in our lab to amplify mRNA from samples of intestinal epithelial cells isolated by laser capture microdissection and from CD4+ and CD8+ T cells isolated by magnetic bead methodologies. We have found that about 30-80 micrograms of biotin-labeled cRNA are synthesized consistently from 50-100 nanograms of total RNA in the starting sample, well more than the 15 ug required for the hybridization reaction. Thus, the efficacy of the processing reactions and the reproducibility of the results make the Two-Cycle Target Labeling Kit a highly recommended system for amplifying and labeling mRNA in samples where the total RNA content is less than 1 microgram.
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Michael D. George, PhD
Research Microbiologist
Dept. of Medical Microbiology and Immunology
University of California, Davis
United States
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