Amino Allyl MessageAmp™ II aRNA Amplification Kit
Amino Allyl MessageAmp™ II aRNA Amplification Kit
Invitrogen™

Amino Allyl MessageAmp™ II aRNA Amplification Kit

The Amino Allyl II MessageAmp™ aRNA Amplification Kit includes reagents for aRNA amplification with amimo allyl NTP incorporation. The kitRead more
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Catalog NumberQuantity
AM175320 Reactions
Catalog number AM1753
Price (USD)
2,920.00
Each
Add to cart
Quantity:
20 Reactions
Price (USD)
2,920.00
Each
Add to cart
The Amino Allyl II MessageAmp™ aRNA Amplification Kit includes reagents for aRNA amplification with amimo allyl NTP incorporation. The kit includes sufficient reagents for 20 reactions. Cy™ dyes are not included.

Improved first- and second-strand cDNA synthesis
Included in the kit is ArrayScript™ RT, a rationally engineered M-MLV reverse transcriptase, which produces equivalent or higher yields of full-length cDNA compared to other enzymes. The second-strand cDNA synthesis reaction has also been optimized to be compatible with the first-strand cDNA products generated with ArrayScript™ RT to allow maximal conversion of first-strand cDNA into full-length double-stranded cDNA templates.

Increased labeling efficiency of your aRNA
The Amino Allyl MessageAmp™ II aRNA Amplification Kit incorporates amino allyl NTP into the aRNA followed by the coupling of its reactive amino group to an NHS ester label (e.g., Cy™ or another dye). This strategy offers several advantages over the direct incorporation of labeled NTPs. Direct incorporation of labeled NTPs is inefficient and results in low yields and low specific activity aRNA and high costs. Unlike lableled NTPs, amino allyl–modified NTPs are incorporated almost as efficiently as unmodified NTPs and are much less expensive than the dye coupled NTPs.
For Research Use Only. Not for use in diagnostic procedures.
Specifications
Includes Label or DyeNo
Labeling MethodIndirect Labeling
Product LineAmbion™, MessageAmp™
Product TypeaRNA Amplification Kit
Quantity20 Reactions
Reverse TranscriptaseArrayScript™
Sample TypePoly(A+) RNA, Total RNA
FormatKit
Unit SizeEach
Contents & Storage
• 60 μl T7 Oligo(dT) Primer (-20°C)
• 22 μl ArrayScript™ Reverse Transcriptase (-20°C)
• 22 μl RNase Inhibitor (-20°C)
• 42 μl 10X First Strand Buffer (-20°C)
• 170 μl dNTP Mix (-20°C)
• 210 μl 10X Second Strand Buffer (-20°C)
• 42 μl DNA Polymerase (-20°C)
• 22 μl RNase H (-20°C)
• 84 μl T7 Enzyme Mix (-20°C)
• 84 μl 10X Reaction Buffer (-20°C)
• 95 μl 50 mM UTP Solution (-20°C)
• 64 μl 50 mM 5-(3-amino allyl)-UTP (-20°C)
• 255 μl ATP, CTP, GTP Mix (-20°C)
• 40 μl Second Round Primers (-20°C)
• 10 μl 1 mg/ml Control RNA (-20°C)
• 1.75 ml Nuclease-free Water (any temperature)
• 400 μl Coupling Buffer (-20°C)
• 440 μl DMSO (-20°C)
• 180 μl 4M Hydroxylamine (-20°C)
• 40 ml Wash buffer (4°C or room temperature)
• 7 ml cDNA Binding Buffer (room temperature)
• 20 ml aRNA Binding Buffer (room temperature)
• 20 aRNA Filter Cartridges (room temperature)
• 40 aRNA Collection Tubes (room temperature)
• 20 cDNA Filter Cartridges + Tubes (room temperature)
• 20 cDNA Elution Tubes (room temperature)
• 10 ml Nuclease-free Water (any temperature)
• 20 Labeled aRNA Filter Cartridges + Tubes (room temperature)
• 20 Labeled aRNA Elution Tubes (room temperature)

Frequently asked questions (FAQs)

What is the typical size range of amplified RNA?

A single round of amplification yields product sizes ranging from 200 bases to 6 kb. The majority of these products are approximately 1.5 kb in length. A second round of amplification will result in shorter products. We recommend using an Agilent 2100 bioanalyzer to visualize these products. Amplification products can be visualized by agarose gel electrophoresis; they will migrate as a smear. Although this data is still useful, it is less informative than bioanalyzer analysis.

Find additional tips, troubleshooting help, and resources within our Epigenetics Support Center.

How do direct and indirect labeling of aRNA differ?

Direct labeling is incorporation of modified NTPs into amplification products during the IVT step of the amplification process. To make aRNA that is labeled with fluorescent dyes, a mixture of dye-modified and unmodified (or unlabeled) nucleotides are typically used in order to obtain an optimal ratio of dye-labeled to unlabeled nucleotide for maximal fluorescence. Usually ~200-400 µM of dye-labeled CTP is used with 1-3 mM unlabeled NTPs. Biotin-modified nucleotides are incorporated fairly well with T7 RNA polymerase. We recommend that you use UTP:biotin-UTP ratios of 1:1 to 3:1. In general, labeled nucleotides are not incorporated as efficiently as unlabeled molecules during amplification, and therefore direct labeling does compromise sample yield. Furthermore, if both Cy5 and Cy3 are used in a direct labeling reaction, Cy5 is not incorporated as well as Cy3, and corrections during data analysis are necessary to adjust for this disparity.

Indirect labeling incorporates amino allyl UTP into amplification products during the IVT, and the amino allyl-modified aRNA produced is then chemically coupled to a detectable moiety such as a fluorescent dye or biotin. This method, though more time-consuming than direct labeling, can result in very highly labeled aRNA because amino allyl-modified UTP is incorporated very efficiently by T7 RNA polymerase.

Find additional tips, troubleshooting help, and resources within our Epigenetics Support Center.

Why is RNA amplification necessary?

Glass microarray analysis experiments typically require 5-20 µg of total RNA per slide for sample labeling and hybridization. Thus, microarray-based gene expression analysis of very small samples [laser capture microdissection (LCM), tissue biopsies, or other clinical samples] is difficult due to the very low amounts of total RNA recovered from the samples. Linear amplification of RNA from small samples produces sufficient quantities of RNA for sample labeling and hybridization. Since the amplification technique is highly reproducible and maintains representation of the gene expression in the original sample, it is recommended for probe synthesis by most manufacturers of commercially available microarrays.

Find additional tips, troubleshooting help, and resources within our Epigenetics Support Center.

How is fold amplification calculated?

RNA amplification using the Van Gelder and Eberwine technique (Van Gelder 1990) utilizes an oligo(dT) primer containing the T7 RNA polymerase promoter for synthesis of first strand cDNA. The poly(A) tail at the end of mRNA sequences serves as the substrate for the binding of these primers. Since mRNA typically constitutes only 1-5% of the total RNA in the cell, only this fraction of the total RNA is amplified. The tissue type, its developmental state, and its health all influence the actual proportion of mRNA in a total RNA sample. Total RNA from brain, testes, and embryonic tissues may contain up to 4% mRNA, while RNA from many other tissues will have only 1% or less mRNA. The RNA isolation method can also influence mRNA content. The generally accepted average value for mRNA content is about 2% of a total RNA sample. When 1 µg of total RNA, 2% or 20 ng of which is mRNA, is amplified 1000-fold, yields of 20 µg aRNA (or cRNA) should be expected. You may observe higher fold amplification when starting with lower amounts of total RNA. This is because, in an in vitro transcription (IVT) reaction, a finite amount of RNA can be synthesized with the fixed amount of NTPs. When starting with less RNA, NTPs do not become limiting until the RNA is amplified beyond the typical 1000-2000 fold amplification levels seen with higher amounts of input RNA.

Find additional tips, troubleshooting help, and resources within our Epigenetics Support Center.

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