Cat. No. / ID: 330411
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.
The RT2 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 RT2 HT First Strand Kit is optimized for real-time PCR-based gene expression analysis using RT2 Profiler PCR Arrays and RT2 qPCR Primer Assays, and can be successfully used with other commercially available gene expression analysis assays.
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.
For cDNA synthesis from 96 samples
Carry out all procedures in a "DNA-free" workspace (see FAQ 2654). Be sure to include any DNase treatment steps in the recommended RNA isolation procedure or treat RNA separately with RNase-free DNase followed by repurification using a spin-column based method. RNeasy Mini Kit can be easily combined with RNase-free DNase. Alternatively, kits like the RNeasy Plus Universal Tissue already include a DNA removal step. Be sure to double both the units of enzyme and the incubation time recommended by the RNase-free DNase manufacturer. To minimize DNA contamination in your RNA preparations, and avoid the need for supplemental DNase treatments, we recommend using the RT2 First Strand Kit, which includes a highly efficient genomic DNA elimination step before reverse transcription.
NOTE: Our Chemistries are not compatible with AMBION's Turbo DNA-Free Kits.