The MessageAmp™ Premier RNA Amplification Kit is the latest innovation for the preparation of RNA samples for microarray analysis. The kit builds upon the linear RNA amplification method and reagents developed for MessageAmp™ II Kits with enhancements that create a simplified workflow, improved performance, and more flexible RNA input requirements. All MessageAmp™ kits employ the proven methodology for RNA sample preparation and labeling based on the original T7 in vitro transcription (IVT) amplification technology, known as the Eberwine or reverse transcription-IVT (RT-IVT) method. The RT-IVT method is considered the gold standard for sample preparation in microarray-based expression profiling. It is well documented in the current scientific literature and was experimentally validated using TaqMan® RT-PCR. MessageAmp™ Premier Kit features:
• Lowest RNA input requirements for a single round of amplification: 20 ng total RNA yields enough biotin-modified aRNA for most microarray formats, with results equivalent to those from samples prepared from significantly more starting material using other RT-IVT kits
• Very flexible RNA input requirements: 20–500 ng total RNA, depending on the tissue or cell type
• Streamlined workflow: depending on the RNA input amounts, only a single day of processing time is required to complete a microarray experiment
• Simplified protocol and streamlined reagents
• Complete kit: everything needed to prepare biotin-modified aRNA is included
• Rigorous quality control that includes GeneChip® analysis
• T7 MEGAscript® IVT delivers up to 50,000-fold amplification
• Single-tube format
• Easy-to-use, high-recovery, magnetic-bead aRNA purification
Using the MessageAmp&trade Premier RNA Amplification Kit
Based on this proven enzymatic amplification and labeling strategy, the MessageAmp™ Premier procedure starts with a simple cDNA synthesis reaction using ArrayScript™ reverse transcriptase. No cleanup step is required after the RT; cDNA is used directly in a high yield IVT reaction using MEGAscript® technology. The IVT is configured to incorporate the modified nucleotide, biotin-UTP, into the aRNA synthesized. (In this protocol, the amplified RNA is referred to as aRNA; in the literature, it is also commonly called cRNA.) Once purified, the aRNA is suitable for use on microarray gene expression analysis platforms designed for biotin-modified antisense RNA samples.
原厂资料：
注意事项：
• 110 µl First Strand Enzyme Mix (-20°C)
• 440 µl First Strand Buffer Mix (-20°C)
• 220 µl Second Strand Enzyme Mix (-20°C)
• 550 µl Second Strand Buffer Mix (-20°C)
• 660 µl T7 Enzyme Mix (-20°C)
• 2 × 1.1 ml T7 Biotin IVT Mix (-20°C)
• 10 µl Control RNA 1 mg/ml (-20°C)
• 1.75 ml Nuclease-free Water (any temperature)
• 6 ml aRNA Binding Buffer Concentrate (room temperature)
• 1.2 ml RNA Binding Beads (4°C)
• 30 ml aRNA Wash Solution Concentrate (room temperature)
• 15 ml aRNA Elution Solution (4°C or room temperature)
• 1 ml 5X Array Fragmentation Buffer (room temperature)
• 2 U-Bottom Plates (room temperature)
• 4 PCR Plates (room temperature)
• 1 Reservoir (room temperature)
• 10 mL Nuclease-free Water (any temperature)
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