RT² First Strand Kit

For cDNA synthesis and genomic DNA elimination in RNA samples for use with RT² Profiler PCR Arrays and RT² lncRNA PCR Arrays

Products

The RT² First Strand Kit is intended for molecular biology applications. This product is not intended for the diagnosis, prevention, or treatment of a disease.

RT² First Strand Kit (12)

Cat. No. / ID: 330401

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)

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RT² First Strand Kit (50)

Cat. No. / ID: 330404

For fifty 20 µl first strand cDNA synthesis reactions; Buffer GE (100 µl), 5x Buffer BC3 (200 µl), RE3 Reverse Transcriptase Mix (100 µl), Control P2 (50 µl), Nuclease-Free Water (1 ml)

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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

Product Details

The RT² First Strand Kit provides a rapid and convenient procedure for efficient first-strand cDNA synthesis and genomic DNA elimination in RNA samples. The synthesized cDNA is ready to use in real-time PCR expression analysis of multiple genes.

Principle

The RT² 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 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 First Strand Kits are 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.

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.

Applications

cDNA synthesized using the RT² First Strand Kit yields excellent results in real-time PCR expression analysis of multiple genes.

Resources

Download Safety Data Sheets for QIAGEN product components.

For pathway-focused gene expression profiling using real-time RT-PCR

Simultaneously profile mRNA, miRNA and lncRNA using a simple, complete workflow

For pathway-focused gene expression analysis

FAQ

You need:

A RT² SYBR Green Mastermix that matches the qPCR instrument in your laboratory;
RT² qPCR Primer Assays for your target genes;
A Housekeeping gene RT² qPCR Primer Assay.

We also recommend using our RT² First Strand Kit for reverse transcription.

FAQ ID -2707

For best results, all RNA samples should be suspended in RNase-free water. Alternatively, RNase-free 1 mM sodium citrate (pH 6.5) or 10 mM Tris buffer (pH 7.0) may be used. Do not use DEPC-treated water, as most DEPC preparations are contaminated with molecules that are inhibitory to reverse transcription and/or PCR. For long-term storage, RNA preps may be stored at -70 ºC in RNase-free water, or the buffers listed above, or precipitated in ethanol or isopropanol. In order to avoid repeated freeze-thaw cycles, it is recommended that frozen RNA samples be stored as multiple, single-use aliquots.

FAQ ID -2659

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.

FAQ ID -2662

RT2 First Strand Kit

For cDNA synthesis and genomic DNA elimination in RNA samples for use with RT2 Profiler PCR Arrays and RT2 lncRNA PCR Arrays