Qiagen, 330401, RT 2 First Strand Kit (12)

For twelve 20 µl first strand cDNA synthesis reactions; Buffer GE (24 µl), 5x Buffer BC3 (48 µl), RE3 Reverse Transcriptase Mix (24 µl), Control P2 (12 µl), Nuclease-Free Water (1 ml)

Features

- Rapid and efficient first-strand cDNA synthesis
- Complete elimination of genomic DNA
- Results with as little as 25 ng total RNA per reaction
- cDNA immediately ready for real-time PCR
- External RNA control to monitor enzyme inhibitors

Principle

The kit includes a proprietary procedure to effectively eliminate contaminating genomic DNA from RNA samples before reverse transcription. Random hexamers and oligo-dT prime reverse transcription in an unbiased manner, and a reverse transcriptase synthesizes cDNA product with optimal yield and length. A built-in external RNA control helps monitor reverse transcription efficiency and test for enzyme inhibitors. The RT 2 First Strand Kits are optimized for real-time PCR-based gene expression analysis using RT 2 Profiler PCR Arrays and RT 2 qPCR Primer Assays, and can be successfully used with other commercially available gene expression analysis assays.

Procedure

The proprietary Buffer GE and sample are mixed and then incubated on a thermal cycler. After addition of the enzyme and additional buffer, the sample is then reverse transcribed using any thermal cycler. The transcribed cDNA is immediately ready for use in downstream applications. We recommend RT² Profiler PCR Arrays or RT² qPCR Primer Assays for further analysis.

RT² First Strand Kits yield results with as little as 25 ng or as much as 5 µg total RNA per reaction. However, the optimal amount of starting material depends on the relative abundance of the transcripts of interest. Lower abundance transcripts require more RNA; higher abundance transcripts require less RNA. Greater amounts of input RNA yield greater number of positive calls (i.e., gene expression detection) in the linear dynamic range of the method.