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

Amino Allyl MessageAmp™ II aRNA Amplification Kit

アミノアリルII MessageAmp™ aRNA増幅キットには、アミノアリルNTP取り込みによるaRNA増幅用試薬が含まれています。本キットには20回の反応分の試薬が含まれています。Cy™色素は含まれていません。改善された第1鎖および第2鎖cDNA合成キットには、合理的に設計されたM-MLV逆転写酵素であるArrayScript™ RTが含まれています。これは詳細を見る
製品番号(カタログ番号)数量
AM175320反応
製品番号(カタログ番号) AM1753
価格(JPY)
436,100
Each
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数量:
20反応
アミノアリルII MessageAmp™ aRNA増幅キットには、アミノアリルNTP取り込みによるaRNA増幅用試薬が含まれています。本キットには20回の反応分の試薬が含まれています。Cy™色素は含まれていません。

改善された第1鎖および第2鎖cDNA合成
キットには、合理的に設計されたM-MLV逆転写酵素であるArrayScript™ RTが含まれています。これは、他の酵素と比較して同等以上の完全長cDNAを生成します。第2鎖cDNA合成反応は、ArrayScript™ RTで生成された第1鎖cDNA製品と互換性を持つように最適化されており、第1鎖cDNAを完全長の二本鎖cDNAテンプレートに最大限に変換できます。

aRNAの標識効率の向上
アミノアリルMessageAmp™ II aRNA増幅キットは、アミノアリルNTPをaRNAに組み込み、その後、反応性アミノ基をNHSエステルラベル(Cy™や他の色素など)に結合します。この戦略は、標識されたNTPの直接組み込みよりもいくつかの利点を提供します。標識されたNTPを直接組み込むことは非効率的で、低収量で特異的活性が低いaRNAと高コストをもたらします。Labeled NTPとは異なり、アミノアリル–修飾NTPは未修飾NTPとほぼ同じ効率で組み込まれており、色素結合NTPよりもはるかに安価です。
研究用にのみ使用できます。診断用には使用いただけません。
仕様
標識または色素を含む不可
標識法間接ラベリング
製品ラインAmbion、MessageAmp
製品タイプaRNA増幅キット
数量20反応
逆転写酵素ArrayScript™
サンプルタイプPoly(A+)RNA、トータルRNA
フォーマットキット
Unit SizeEach
組成および保存条件
•T7 Oligo(dT)プライマー60 µL(-20℃)
•ArrayScript™逆転写酵素22 µL(-20℃)
•RNase阻害剤22 µL(-20℃)
•10X第一鎖バッファー42 µL(-20℃)
•dNTPミックス170 µL(-20℃)
•10X第二鎖バッファー210 µL(-20℃)
•DNAポリメラーゼ42 µL(-20℃)
•RNase H 22 µL(-20℃)
•T7酵素ミックス84 µL(-20℃)
•10X反応バッファー84 µL(-20℃)
•50 mM UTP溶液95 µL(-20℃)
•50 mM 5-(3-アミノアリル)-UTP 64 µL(-20℃)
•ATP、CTP、GTPミックス255 µL(-20℃)
•第2ラウンドプライマー40 µL(-20℃)
•1 mg/mLコントロールRNA 10 µL(-20℃)
•ヌクレアーゼフリー水1.75 mL(任意の温度)
•結合バッファー400 µL(-20℃)
•DMSO 440 µL(-20℃)
•4 Mヒドロキシラミン180 µL(-20℃)
•洗浄バッファー40 mL(4℃または室温)
•cDNA結合バッファー7 mL(室温)
•aRNA結合バッファー20 mL(室温)
•aRNAフィルターカートリッジ20個(室温)
•aRNA収集チューブ40本(室温)
•cDNAフィルターカートリッジ+チューブ20セット(室温)
•cDNA溶出チューブ20本(室温)
•ヌクレアーゼフリー水10 mL(任意の温度)
•ラベル付きaRNAフィルターカートリッジ+チューブ20セット(室温)
•ラベル付きaRNA溶出チューブ20本(室温)

よくあるご質問(FAQ)

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.