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Transcription reactions with MEGAscript® High Yield Transcription Kits use novel reaction conditions and Ambion's patented, high yield technology that allow the synthesis of 10 to 50 times the amount of RNA produced by conventional transcription reactions. Each DNA template is copied hundreds of times, allowing the production of milligram quantities of RNA from a single kit.

The yield of MEGAscript Kit reactions depends both on the polymerase and the template being transcribed, but a typical 20 µl MEGAscript reaction with 1 µg of the pTRI-Xef-1alpha control template will yield over 100 µg of transcript. The versatility of the MEGAscript kit allows the manipulation of the reactions to include specialized reagents such as modified nucleotides, cap analog or additional polymerase. The RNA synthesized from a MEGAscript reaction is suitable for many applications including in vitro translation, microinjection, ribozyme studies, RNA structural analysis, nonisotopic probes, low specific activity probes, RNAi analysis, RNA vaccines and aRNA amplification.

The MEGAscript Kit can be used to overcome a primary limitation of gene profiling with gene arrays. Gene profiling using gene arrays requires up to 100 µg of total RNA or 5 µg of poly(A) RNA for each hybridization. It is often impossible to obtain enough RNA for this application from small samples such as cancer cell lines, excised surgical tissues, or cells obtained from Laser Capture Microdissection. MEGAscript has been used extensively to solve this problem through the amplification of low-abundance messages from samples where RNA is limiting (see citations at right). RNA is isolated from the tissue, reverse transcribed, and amplified via antisense RNA amplification using the MEGAscript Kit.

The MessageAmp™ II Kit, developed especially for the amplification of cRNA prior to array analysis, is powered by MEGAscript technology. MessageAmp II can amplify as little as 100 ng of input RNA in a single round for GeneChip® analysis. A rationally engineered MMLV RT, ArrayScript™, is provided in the kit to maximize yields of full-length cDNA. These features result in high labeling efficiency, yielding consistently high percent present-calls and low 3'/5' ratios in GeneChip analysis. This procedure is compatible with biotin, cyanine dye (Perkin Elmer), and Cy™Dye (Amersham Biosciences) labeling either during or after cRNA amplification.

To purify in vitro transcription products from free nucleotides, buffer components, and enzymes, Ambion recommends using the MEGAclear™ Purification Kit. MEGAclear is a fast, convenient, solid phase clean up method that is ideal for purifying large scale transcription reactions.

In some situations, it may be necessary to completely remove the plasmid DNA template after RNA synthesis. For example, it is desirable to eliminate any residual DNA before using the nascent RNA as a standard for RT-PCR or in microinjection experiments. Ambion strongly recommends TURBO DNase™ for this task. TURBO DNase is 63-fold more effective than standard DNase I in clearing DNA from in vitro transcription reactions, such as MEGAscript or mMessage mMachine™, as measured by real-time RT-PCR. http://www.ambion.com/catalog/CatNum.php?AM1334