First Cycle, second strand cDNA synthesis

Second Cycle, first strand cDNA synthesis

Invitrogen RETROscript Reverse Transcription Kit 40 ..

Important Note: Each RT² PreAMP Primer Mix is PCR Array-specific and can only be used for the specified RT² Profiler PCR Arrays. The first strand synthesis components and the RT² PreAMP reagents in this kit have been optimized to maximize the sensitivity of our RT² Profiler PCR Arrays.

Do you have any suggestions for incorporating the Two-Cycle cDNA Synthesis Kit into our routine GeneChip microarray analysis?

Two-Cycle cDNA Synthesis Kit - Affymetrix - Help

To avoid DNA contaminations it is beneficial to include a DNAse step before using the RNA for cDNA synthesis. See below the description of the DNA free kit purchased from .

The kit includes a proprietary procedure to effectively eliminate contaminating genomic DNA from RNA samples before reverse transcription.

Total RNA from RBCs was purified using reagents provided in the PAXgene Blood RNA Kit (, Germany) and tested with RNA LabChip Kit by 2100 Bioanalyzer (, USA). cDNA was synthesised from 5µg total RNA using the SuperScript Double-Stranded cDNA Synthesis Kit (, USA) and purified according to the manufactures' instruction (GeneChip Sample Cleanup Module, , USA). Biotin-labeled cRNA was synthesized using the BioArray HighYield RNA Transcript Labeling Kit (, USA) and purified using GeneChip Sample Cleanup Module (, USA). Yield and size distribution of the labeled transcripts were determined with NanoDrop (, Germany) and 2100 Bioanalyzer (, USA). Fragmentation was carried out using the fragmentation buffer from GeneChip Sample Cleanup Module (, USA).

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Affymetrix - Help - FAQ - Two-Cycle cDNA Synthesis Kit …

Genome-wide gene expression analysis has become a very powerful routine tool for the study of distinct differentiation states. However, the examination of total populations of cells that contain high levels of heterogeneity, such as the total CD8+ T cell population during an immune response, is limited because that complexity hampers accurate interpretation. The gene expression signatures from populations represent the average of all cells within the populations, which will smooth out large expression changes within small subpopulations and virtually eliminate any small changes. However, small expression changes within a minor subpopulation, such as antigen-specific CD8+ T cells responding to an infection, can have relevant biological consequences. Although very limited amounts of RNA can be isolated from small subpopulations of cells, there are now methods to synthesize and amplify cDNA from this limited RNA in sufficient quantities needed for microarray analysis. Here, we describe a complete protocol to extract RNA from small numbers of cells, synthesize cDNA from that RNA, and amplify that cDNA in an unbiased method. This protocol is a useful tool for the study of genome-wide expression signatures from many of the subpopulations that are numerically small but important in immune responses and homeostasis.

cDNA made using Stratagene ZAP-cDNA synthesis kit; ..

The Marathon cDNA Amplification Kit method employs a specially-designed adaptor that significantly reduces background and permits both 5'- and 3'-RACE reactions (1, 2) to be performed using the same template. Marathon cDNA amplification can be used to quickly characterize multiple RNAs identified by expressed sequence tags (ESTs), differential display, RNA fingerprinting, or cDNA subtraction. Marathon cDNA synthesis begins with poly A+ RNA and a modified lock-docking oligo(dT) primer that contains two degenerate nucleotides at the 3' end. These nucleotides position the primer at the beginning of the poly A+ tail, eliminating the 3' heterogeneity inherent with conventional oligo(dT) priming. Following cDNA synthesis, blunt ends are created and the Marathon Adaptor is ligated to both ends of the double-stranded cDNA.