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Luna Universal Probe One-Step RT-qPCR Kit

Luna Universal Probe One-Step RT-qPCR Kitカタログ番号:E3006

カタログ番号

サイズ

濃度

価格

保存温度

E3006S 200 rxns 2X ¥23,500 -20C
E3006L 500 rxns 2X ¥52,800 -20C
E3006X 1,000 rxns 2X ¥92,500 -20C
E3006E 2,500 rxns 2X ¥204,000 -20C

製品カテゴリ>グループ

  • リアルタイム PCR>1-ステップ RT-qPCR (プローブ)

特徴

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*新型コロナウイルス RNA の検出に関する最新論文とアプリケーションノートを公開しました(2020年4月9日)
 
特長:

 ・RNA 定量用 1-ステップ RT-qPCR キット

 ・プローブ・アッセイ用試薬

 ・Hot Start DNAポリメラーゼと Warm Start 逆転写酵素の採用により、非特異的反応が抑制され、室温でのセットアップが可能

 ・ピペッティング、分注ミスを低減する青色の 2X マスターミックス*

 ・補正用ダイが含まれており、ROX の添加は不要

   *青色トラッキング・ダイは qPCR 反応やシグナルに影響を及ぼすことはありません。
 
 
図1:ルナ 1 ステップ RT-qPCR キット は高い感度と再現性を示す
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0.1 pg - 1 μg の 広範囲のトータル RNA から高い再現性で 1 ステップ RT-qPCR ができる。ヒト GAPD をターゲットとしてN=8で RT-qPCR をおこなった (N=8) 。逆転写反応は 55 ℃、10分間で実施した。

 
 
図2:RNA サンプルを問わず、正確に検出・定量が可能、さらに複数ターゲットの同時検出も可能
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Luna Universal Probe One-Step RT-qPCR Kit を用いたワンステップ RT-qPCR により 3 種類のターゲットを同時に増幅したところ、いずれも良好に増幅・定量できることが示された。1 pg–1 µg の Jurkatトータル RNA をテンプレートとした。ターゲットはヒト GAPDH、Ribosomal Protein L32g、Pl3-KinasRelated Kinase SMG1 である。それぞれ異なるコピー数であるため、異なるプライマー濃度を使用した(低コピーの SMG1 は 0.4 µM、高コピーの GAPDH と L32gは 0.2 µM)。各ターゲットのプローブには異なる蛍光を使用した(FAM、HEX、Cy5)。

 

 
 
図3:高い特異性と安定した性能
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Jurkat ゲノム DNA または cDNA をテンプレートとして、各社製品を用いて、様々なコピー数、長さ、GC 含量の 7 領域を対象に RT-qPCR を行った結果、NEB のルナが最も高い特異性(検出率)を示した。各社試薬の推奨条件に従い、2 名が個別に実験を行った。増幅効率、感度(低インプットの検出)、非特異的増幅の有無について結果を評価した(ΔCq= 最低インプットの平均 Cq – 非テンプレートコントロールの平均 Cq)。また一貫性と再現性、増幅曲線の信頼性を Quality Score として評価に加えた。各社製品の詳細は以下のとおり: Quanta, qScript™ XLT 1-Step RT-qPCR ToughMix®; ABI, TaqMan® RNA-to-Ct 1-Step Kit; QIAGEN, QuantiFast® Probe RT-PCR Kit; Bio-Rad, iTaq™ Universal Probes One-Step Kit; Promega®, GoTaq® Probe 1-Step RT-qPCR System。

 
“dots in boxes(2番目の図)”の詳細は NEB TV Japan(日本語字幕付動画)を参照。

製品内容

構成:

・Nuclease-free Water

・Luna WarmStart RT Enzyme Mix

・Luna Universal Probe One-Step Reaction Mix


保存温度:

-20℃

NEB TV Japan:リアルタイムPCR

image   image  

 リアルタイム PCR の原理

 

 Dots in Boxes 法による

 qPCR データの可視化と評価

 

 

NEB TV Japan 一覧(日本語字幕付)

メモ

1. Primer Design

The use of qPCR primer design software (e.g., Primer3) maximizes the likelihood of amplification success while minimizing nonspecific amplification and primer dimers. Targets with balanced GC/AT content (40–60%) tend to amplify most efficiently. Where possible, enter sufficient sequence around the area of interest to permit robust primer design and use search criteria that permit cross-reference against relevant sequence databases (to avoid potential off-target amplification). It is advisable to design primers across known RNA splicing sites in order to prevent amplification from genomic DNA.


2. Primer and Probe Concentrations
For most targets, a final concentration of 400 nM (each primer) will provide optimum performance. If needed, primer concentrations can be optimized between 100–900 nM. Probe should be included at 200 nM for best results. Probe concentration can be optimized in the range of 100–500 nM.

3. Multiplexing
When determining which fluorophores to include in a multiplex reaction, be sure to choose compatible reporter dyes and quenchers (e.g., those that can be accommodated by the chosen real-time instrument with minimal overlap in fluorescence spectra). For ROX-dependent instruments, avoid ROX-labeled probes. Include 400 nM of forward and reverse primers and 200 nM probe for each target to be detected in the reaction. For targets that differ significantly in abundance, use of a lower primer concentration (e.g. 200 nM) for the more abundant target(s) is recommended. Adjust concentrations if necessary based on performance (primer 100–900 nM, probe 100–500 nM). When loading a qPCR protocol onto the real-time instrument, be sure to select the appropriate optical channels, as some instruments have a single channel recording mode that would prevent multiplex data collection and analysis. The functionality of the primer and probe sets should be tested individually before attempting a multiplex reaction.

4. Amplicon Length
To ensure successful and consistent qPCR results, it is important to maximize PCR efficiency. An important aspect of this is the design of short PCR amplicons (typically 70–200 bp). Some optimization may be required for targets that exceed that range.

5.Template Preparation and Concentration
Luna RT-qPCR is compatible with RNA samples prepared through typical nucleic acid purification methods. Prepared RNA should be stored in an EDTA-containing buffer (e.g., 1X TE) for long-term stability, and dilutions should be freshly prepared for a qPCR experiment in either TE or water. Note that the quality of RNA templates can greatly affect RT-qPCR efficiency. RNA should be handled with appropriate precautions to prevent RNase or DNase contamination. Use of nuclease-free water (provided) is strongly recommended. Where useful, RNA may be treated with DNase I to remove contaminating genomic DNA.
Generally, a useful concentration of standard and unknown material will be in the range of 108 copies to 10 copies. Note that for dilutions in the single-copy range, some samples will contain multiple copies and some will have none, as defined by the Poisson distribution. For total RNA, Luna One-Step Kits can provide linear quantitation over an 8-order input range of 1 μg – 0.1 pg. For most targets, a standard input range of 100 ng – 10 pg total RNA is recommended. For purified mRNA, input of ≤ 100 ng is recommended. For in vitro-transcribed RNA, input of ≤ 109 copies is recommended.

6.ROX Reference Dye
Some real-time instruments recommend the use of a passive reference dye (typically ROX) to overcome well-to-well variations that could be caused by machine limitations such as “edge effect”, bubbles, small differences in volume, and autofluorescence from dust or particulates in the reaction. However, ROX normalization does little to the variations caused by pipetting errors of templates/primers, heterogeneous mixing, and evaporation/condensation issues.

A universal passive reference dye is included in the following Luna® qPCR products: Luna Universal qPCR Master Mix (NEB #M3003), Luna Universal Probe qPCR Master Mix (NEB #M3004), Luna Universal One-Step RT-qPCR Kit (NEB #E3005), and Luna Universal Probe One-Step RT-qPCR Kit (NEB #E3006). These products support broad instrument compatibility (High-ROX, Low-ROX, ROX-independent) so no additional ROX is required for normalization.

The Luna Probe One-Step RT-qPCR Kit (No ROX) (E3007) contains no reference dye and is compatible with any instrument that does not require ROX. If ROX normalization is needed, ROX can be added. Please refer to instrument manufacturer’s instructions for greater details.

7. Carryover Contamination Prevention
RT-qPCR is an extremely sensitive method, and contamination in new RT-qPCR assays with products from previous amplification reactions can cause a variety of issues, such as false positive results and a decrease in sensitivity. The best way to prevent this “carryover” contamination is to practice good laboratory procedures and avoid opening the reaction vessel post amplification. However, to accommodate situations where additional anti-contamination measures are desired, Luna qPCR mixes contains a mixture of dUTP/dTTP that results in the incorporation of dU into the DNA product during amplification. Pretreatment of qPCR/RT-qPCR experiments with uracil DNA glycosylase (UDG) will eliminate previously-amplified uracil-containing products by excising the uracil base to produce a non-amplifiable DNA product. The use of a thermolabile UDG is important, as complete inactivation of the UDG is required to prevent destruction of newly synthesized qPCR products.

To enable carryover prevention, 0.025 units/μl Antarctic Thermolabile UDG (NEB #M0372) should be added to the reaction mix. To maximize elimination of contaminating products, set up the qPCR experiments at room temperature or include a 10 minute incubation step at 25°C before the initial denaturation step.

8. Reaction Setup and Cycling Conditions
Due to dual hot-start feature of Luna One-Step Kits, it is not necessary to set up reactions on ice or preheat the thermocycler prior to use.
For 96-well plates, a final reaction volume of 20 μl is recommended.
For 384-well plates, a final reaction volume of 10 μl is recommended.
When programming instrument cycling conditions, ensure a plate read is included at the end of the extension step, and a denaturation (melt) curve after cycling is complete to analyze product specificity.
Amplification for 40 cycles is sufficient for most applications, but for very low input samples 45 cycles may be used.

関連製品

Luna Universal One-Step RT-qPCR Kit (1 ステップ RT-qPCR キット、ダイ)

Luna Universal Probe qPCR Master Mix (qPCR マスターミックス、プローブ)

Luna Universal qPCR Master Mix (qPCR マスターミックス、ダイ)

Antarctic Thermolabile UDG (キャリーオーバー防止用)