Invitrogen™ Illumina™ TotalPrep™-96 RNA Amplification Kit

Invitrogen™ Illumina™ TotalPrep™-96 RNA Amplification Kit

The TotalPrep™-96 RNA Amplification Kit is a complete system for generating biotinylated, amplified RNA for hybridization with Illumina Sentrix® arrays. The kit includes sufficient reagents for 96 reactions.

The TotalPrep™-96 RNA Amplification Kit incorporates extensive improvements to the in vitro transcription reaction (IVT) over the previous TotalPrep™ Kit. The IVT reaction has been optimized for the most effective biotin labeling. An NTP mix containing biotinylated UTP has been added to the kit, allowing ease of use and optimal labeling. These modifications to the kit have significantly increased sensitivity, providing increased Percent Present calls and single-round amplification with just 50 ng of input RNA. The kit is based on the RNA amplification protocol developed in the laboratory of James Eberwine (Van Gelder et al. 1990). The procedure consists of reverse transcription with an oligo(dT) primer bearing a T7 promoter using ArrayScript™, a reverse transcriptase (RT) engineered to produce higher yields of first strand cDNA than wild type enzymes. ArrayScript™ catalyzes the synthesis of virtually full-length cDNA, which is the best way to ensure production of reproducible microarray samples. The cDNA then undergoes second strand synthesis and magnetic-based cleanup to become a template for in vitro transcription with T7 RNA Polymerase. To maximize cRNA yield, MEGAscript® in vitro transcription (IVT) technology, along with biotin UTP (provided in the kit), is used to generate hundreds to thousands of biotinylated, antisense RNA copies of each mRNA in a sample.

Features

• Perform amplification from just 50 ng of input RNA
• Developed for expression profiling using the Sentrix® BeadChips
• Bio-16-UTP now included in optimized NTP mix, reducing set-up time and maximizing labeling

Contents

• T7 Oligo(dT) Primer (-20°C): 110 µl 
• ArrayScript™ Reverse Transcriptase (-20°C): 110 µl 
• 10X First Strand Buffer (-20°C): 220 µl 
• dNTP Mix (-20°C): 880 µl 
• RNase Inhibitor (-20°C): 110 µl 
• RNase H (-20°C): 110 µl 
• 10X Second Strand Buffer (-20°C): 1.1 ml 
• DNA Polymerase (-20°C): 220 µl 
• T7 Enzyme Mix (-20°C): 275 µl 
• Biotin-NTP Mix (-20°C): 275 µl 
• Control RNA (-20°C): 10 µl 
• cDNAPure (4°C): 20 ml 
• RNA Binding Beads (4°C): 1.1 ml 
• cDNA Wash Buffer Concentrate (room temperature): 40 ml 
• cRNA Binding Buffer Concentrate (room temperature): 5.5 ml 
• Bead Resuspension Solution (room temperature): 450 µl 
• cRNA Wash Solution Concentrate (room temperature): 25 ml 
• cRNA Elution Buffer (room temperature): 10 ml 
• U-Bottom Plates (room temperature): 8 Plates
• PCR Plates (room temperature): 8 Plates
• Nuclease-free Water (any temperature): 20 ml