Illumina® TotalPrep™-96 RNA Amplification Kit

Catalog number: 4393543

Ambion™  Related applications: Microarray Analysis

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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. Features of the TotalPrep™-96 RNA Amplification Kit:

• 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

Using the TotalPrep™-96 RNA Amplification Kit
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.

Accessory product
A 96-well magnetic-ring stand is required to use this kit. The 96-well Magnetic-Ring Stand (Cat. No. AM10050) is recommended and designed for paramagnetic bead precipitation from standard 96-well U-bottom microplates. The Magnetic-Ring Stand pellets the magnetic beads in a circle shape, enabling easy removal of supernatant by a robot or multichannel pipettors.
For Research Use Only. Not for use in diagnostic procedures.


System: Illumina Sentrix®
Product Size: 96 reactions
Labeling Method: Direct Labeling
Includes Label or Dye: Yes
Reverse Transcriptase: ArrayScript™ Reverse Transcriptase
Sample Type (Specific): Total RNA, Poly(A+) RNA

Contents & storage

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


Manuals & protocols

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