White Paper, GeneChip® 3 ' IVT Express Kit

The GeneChip 3’ IVT Express Kit is the newest option for generating target for Affymetrix 3’ expression arrays. The 3’ IVT Express Kit utilizes the same basic chemistry as GeneChip One-Cycle Target Labeling and Control Reagents, but employs a simplified workflow and optimized reagents that enable lower RNA input and increased ease of use. In this white paper, we describe the technical performance of the 3’ IVT Express Kit and compare array results with One-Cycle Reagents. Introduction Affymetrix 3’ expression arrays have probe sets targeted to the 3’ end of the transcript. Target for these arrays is prepared using amplification and labeling methodologies that generate labeled target initially primed from the poly-A tail of the transcript. First-strand cDNA is synthesized using an oligo-dT primer with an attached T7 promoter sequence. After making the complementary second strand, the double-stranded cDNA molecule is amplified in a linear fashion by in vitro transcription (IVT) using T7 RNA polymerase. As the aRNA (amplified RNA, also commonly referred to as cRNA) is being made, a biotinylated nucleotide analog is incorporated and serves as a label for the message. After fragmentation, this labeled anti-sense aRNA is hybridized to the array. We have developed a new kit that utilizes this same basic strategy for generating target for hybridization to GeneChip 3’ expression arrays. The 3’ IVT Express Kit uses optimized reagents that dramatically reduce the total RNA input requirements. For most RNA types, 50 to 100 ng of total RNA will generate sufficient amounts of labeled target for the largest-format cartridge array. Because of the low total RNA input requirements, the 3’ IVT Express Kit is intended to replace the One-Cycle and Two-Cycle Target Labeling and Control Reagents. In addition, the 3’ IVT Express Kit provides a simplified workflow and includes pre-made master mixes that decrease hands-on time (see Figure 1). Those using higher input amounts (100 to 500 ng) can complete the target preparation protocol in a single day. Compared to One-Cycle Reagents, the cDNA cleanup step has been removed and all purification steps now use magnetic beads instead of spin columns for enhanced recovery. The kit contains all of the reagents needed to generate labeled aRNA target and also includes a control RNA sample, poly-A RNA and hybridization controls, and the plastic consumables needed to run the assay.