Description
The QuantiTect Whole Transcriptome Kit enables the preamplification and reverse transcription of limited amounts of RNA to high yields of cDNA for unlimited gene expression analysis using real-time PCR. An optimized protocol ensures a fast and easy procedure. The kit consists of a complete set of enzymes and buffers for whole transcriptome amplification (WTA). Innovative modification of Phi 29 polymerase technology allows yields of up to 40 μg cDNA from as little as 1 ng RNA. The uniquely high processivity of this polymerase guarantees the generation of cDNA containing uniformly amplified targets to ensure reliable gene expression analysis in real-time PCR.
The QuantiTect Whole Transcriptome Kit provides highly uniform amplification of all transcripts, which is essential for reliable gene expression analysis. All mRNA transcripts are amplified with equal representation at both 5' and 3' regions (see figure "Equal amplification of 5' and 3' regions"). Preservation of the transcript profile is demonstrated in the figure "Preservation of transcript profile", where WTA-amplified cDNA prepared using the QuantiTect Whole Transcriptome Kit is compared with nonamplified cDNA prepared using a reverse transcriptase. High and reproducible cDNA yields of up to 40 μg can be achieved (see figure "Reproducible cDNA yields"). This allows unlimited real-time PCR analysis with highly reproducible C T values (see table and figure "Reliable real-time PCR analysis"). In addition, since cDNA is more stable than RNA, it can be safely archived for future studies. High yields of cDNA for real-time PCR analysis Starting material Amplification time Typical yield of amplified product Number of real-time PCR analyses* 10 ng RNA 2 hours Up to 10 µg cDNA 1000 10 ng RNA 8 hours Up to 40 µg cDNA 4000 * Number of real-time PCR analyses possible using cDNA from a single QuantiTect whole transcriptome amplification (WTA) reaction. Without WTA, only 1 reliable real-time RT-PCR analysis is possible from 10 ng RNA.
Gene expression profiling can be limited by the small amount of biological sample available. By using the QuantiTect Whole Transcriptome Kit, unlimited real-time PCR analyses of small and precious samples are possible. The QuantiTect Whole Transcriptome Kit is optimized for whole transcriptome amplification from as little as 1 ng RNA, or RNA corresponding to about 50 cells. Even lower amounts of RNA can be used, depending on the quality of the RNA and the abundance of the transcript of interest. The QuantiTect Whole Transcriptome Kit contains reverse transcriptase, DNA polymerase, and optimized buffers and reagents for the amplification of all transcripts within an RNA sample. The kit integrates cDNA synthesis with the proven quality of REPLI-g technology for nonbiased sequence amplification. REPLI-g amplification uses Multiple Displacement Amplification (MDA) technology, which carries out isothermal sequence amplification using a uniquely processive DNA polymerase (see figure "Whole transcriptome amplification"). This technology ensures unbiased amplification of all transcript regions, even of 5' ends, providing unlimited cDNA highly suited for real-time PCR.
Unlimited cDNA representing the entire transcriptome is prepared from total RNA in a simple 3-step protocol (see flowchart "Procedure"). RNA is first reverse transcribed into cDNA using an optimized reverse transcription mix containing T-Script Enzyme with random and oligo-dT primers. The synthesized cDNA is ligated using a high-efficiency ligation mix, and then amplified using an amplification mix based on proven REPLI-g technology.
