Qiagen, 330411, RT ² HT First Strand Kit (96)

RT 2 HT First Strand Kit

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 is immediately ready for real-time PCR
- External RNA control to monitor enzyme inhibitors

Product Details

The RT² HT First Strand Kit provides a rapid and convenient procedure for efficient first-strand cDNA synthesis and genomic DNA elimination in RNA samples. It enables simultaneous and easy parallel processing of 96 RNA samples for reverse transcription. The synthesized cDNA is ready to use in real-time PCR expression analysis of multiple genes.

Principle

The RT 2 HT First Strand Kit provides a rapid and convenient procedure for efficient cDNA synthesis from mRNA for use in gene expression assays.

The kit includes a proprietary procedure to effectively eliminate contaminating genomic DNA from RNA samples before reverse transcription. Random hexamers and oligo-dT primers 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 HT First Strand Kit is 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

Each sample is mixed with the proprietary Buffer GE2 in separate wells of a 96-well plate. The plate is sealed using the sealing film provided and incubated on a thermal cycler. After addition of the master mix (BC4 Reverse Transcriptase Mix), 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.

The RT² HT First Strand Kit yields 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.