QuantiTect Probe RT-PCR Kit
For one-step qRT-PCR using sequence-specific probes for gene expression analysis
- Highly sensitive detection of low-copy targets
- Accurate quantification over several logs of template
- Use of any sequence-specific probe on any real-time cycler
- No need to optimize reaction and cycling conditions
QuantiTect Probe RT-PCR Kits enable sensitive quantification of RNA targets by real-time one-step PCR using sequence-specific probes. The combination of a hot start and a unique PCR buffer system in the ready-to-use master mix ensures highly sensitive qRT-PCR on any real-time cycler without the need for optimization. The dNTP mix includes dUTP, allowing optional treatment with UNG. For convenience, the master mix can be stored at 2–8°C.
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QuantiTect Probe RT-PCR Kit (200)
For 200 x 50 µl reactions: 3 x 1.7 ml 2x QuantiTect Probe RT-PCR Master Mix, 100 µl QuantiTect RT Mix, 2 x 2 ml RNase-Free Water
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204443
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QuantiTect Probe RT-PCR Kit (1000)
For 1000 x 50 µl reactions: 25 ml 2x QuantiTect Probe RT-PCR Master Mix, 0.5 ml QuantiTect RT Mix, 20 ml RNase-Free Water
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204445
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QuantiTect Probe RT-PCR Kit は分子生物学実験用です。疾病の診断、治療または予防の目的には使用することはできません。
See trademarks.
One-step RT-PCR.|Highly specific amplification.|High sensitivity and efficiency, and a wide dynamic range.|One-step RT-PCR with comparable performance to two-step RT-PCR - A.|One-step RT-PCR with comparable performance to two-step RT-PCR - B.|Specific primer annealing.|
The QuantiTect Probe RT-PCR Kit overcomes the need for optimization of reaction conditions, which can be tedious and time-consuming. Simply add primers, probe, and RNA template to the ready-to-use RT-PCR master mix, and start the reaction on any real-time cycler (see also table "Components of the QuantiTect Probe RT-PCR Kit").|In comparison with other DNA polymerases, only HotStarTaq DNA Polymerase in combination with a unique buffer specifically amplified a 497 bp fragment (from 50 copies of an HIV-pol-gene construct in a background of 1 µg human genomic DNA). M: Markers.|Tenfold serial dilutions of HeLa cell RNA (100 ng to 1 pg) were analyzed in duplicate on the ABI PRISM 7900 using the QuantiTect Probe RT-PCR Kit with primers and a HEX labeled probe specific for human 28S rRNA. High PCR efficiency over 6 logs of template dilution was achieved.|Tenfold serial dilutions of cDNA (100 ng to 1 pg) prepared from leukocytes were analyzed in duplicate on the Mx3005 using primers and a FAM labeled probe specific for IL1R2 (interleukin 1 receptor, type II). Two-step RT-PCR was performed using the QuantiTect Probe PCR Kit (PCR efficiency: 101%).|Tenfold serial dilutions of RNA (100 ng to 1 pg) prepared from leukocytes were analyzed in duplicate on the Mx3005 using primers and a FAM labeled probe specific for ILR2 (interleukin 1 receptor, type II). One-step RT-PCR was performed using the QuantiTect Probe RT-PCR Kit (PCR efficiency: 104%).|A balanced combination of KCl and (NH4)2SO4 promotes specific annealing of primers and probes to the PCR template. K+ binds to phosphate groups on double-stranded DNA, stabilizing annealing of primers and probes. NH4+ destabilizes weak hydrogen bonds between mismatched bases.|
Principle
QuantiTect Probe RT-PCR Kits contain an optimized, ready-to-use master mix for highly specific and sensitive real-time quantification of RNA targets using sequence-specific probes. The kits are designed for use with all types of sequence-specific probes, including hydrolysis probes (e.g., TaqMan® and other dual-labeled probes), FRET probes, and Molecular Beacons. QuantiTect Probe RT-PCR Kits contain a unique PCR buffer that contains a balanced combination of K+ and NH4+ ions, which promote specific primer annealing, enabling high PCR specificity and sensitivity (see figure "Specific primer annealing"). In addition, an optimized mix of reverse transcriptases enables cDNA synthesis from a wide range of RNA template amounts, while HotStarTaq DNA Polymerase provides a stringent hot start, preventing the formation of nonspecific products.
QuantiTect Probe RT-PCR Master Mix also contains dUTP, enabling pretreatment with uracil-N-glycosylase (UNG) prior to starting PCR, which ensures that any contaminating PCR products do not affect subsequent PCR reactions.
| HotStarTaq DNA Polymerase |
15 min activation at 95ºC |
Set-up of qPCR reactions at room temperature |
| QuantiTect Probe RT-PCR Buffer |
Balanced combination of NH4+ and K+ ions |
Specific primer annealing ensures reliable PCR results |
| dNTP mix |
Includes dUTP, which partially replaces dTTP and enables optional UNG treatment of reactions |
Eliminates contamination from carryover of PCR products by optional UNG treatment |
| ROX dye |
For normalization of fluorescent signals on Applied Biosystems and, optionally, Agilent instruments |
Precise quantification on cyclers that require ROX dye. Does not interfere with reactions on other real-time cyclers |
| Omniscript and Sensiscript Reverse Transcriptases |
Special blend of enzymes with high affinity for RNA |
RNA can be transcribed, even through complex secondary structures |
Procedure
The QuantiTect Probe RT-PCR Kit overcomes the need for optimization of reaction conditions, which can be tedious and time-consuming. Simply add primers, probe, and RNA template to the ready-to-use RT-PCR master mix, and start the reaction (see flowchart "One-step RT-PCR"). Follow the protocol in the handbook to get fast and reliable results on any real-time cycler. If required, reactions can be pretreated with uracil-N-glycosylase (UNG) to eliminate carryover of PCR products from previous reactions.
Applications
QuantiTect Probe RT-PCR Kits can be used for gene expression analysis of RNA targets on any real-time cycler. This includes instruments from Applied Biosystems, Bio-Rad, Cepheid, Eppendorf, Roche, and Agilent. For the Rotor-Gene Q and other Rotor-Gene cyclers, we recommend using the Rotor-Gene Probe RT-PCR Kit, which has been specially developed for fast cycling on these instruments.
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Feature
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Specifications
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Applications
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Real-time quantification of RNA targets
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Reaction type
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One-step RT-PCR
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Real-time or endpoint
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Real-time
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Sample/target type
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RNA targets
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Single or multiplex
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Single
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SYBR Green I or sequence-specific probes
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Sequence-specific probes
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Thermal cycler
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All real-time cyclers (e.g. LC, RG, ABI)
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With or without ROX
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With ROX
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FAQ ID -1056
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FAQ ID -784
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For quantitative, real-time one-step RT-PCR using sequence-specific probes
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詳細を表示
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配列特異的プローブを用いたリアルタイム定量1ステップRT-PCR
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詳細を表示
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Download Safety Data Sheets for QIAGEN product components.
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画像
1ステップRT-PCR
QuantiTect Probe RT-PCR Kit は面倒で時間のかかる反応条件の至適化が不要。プライマー、プローブ、RNA テンプレートを即使用可能なRT-PCRマスターミックスに添加するだけで、どのリアルタイムサイクラーでも反応を開始することができる(表"QuantiTect Probe RT-PCR Kit の成分"も参照)。
特異性の高い増幅
他社のDNAポリメラーゼと比較した結果、ユニークなバッファーと組み合わせたHotStarTaq DNA Polymeraseのみが497 bp フラグメントを特異的に増幅させた(1 µgのヒトゲノムDNAに添加した50 コピーのHIV-pol-遺伝子コンストラクトからのフラグメント)。M:マーカー。
幅広いダイナミックレンジにわたり効率的で感度の高い定量を実現
ヒト28S rRNAに特異的なプライマーおよび HEX標識プローブとQuantiTect Probe RT-PCR Kitを用いて、HeLa細胞RNAの10倍連続希釈液(100 ng~1 pg)をABI PRISM 7900 でduplicateで解析した。テンプレートを106倍希釈した溶液で高いPCR効率が得られた。
2ステップRT-PCRのパフォーマンスと同等の1ステップRT-PCR - A
IL1R2(interleukin 1 receptor, type II)に特異的なプライマーおよび FAM標識プローブを用いて、白血球から調製したcDNA(100 ng~1 pg)の10倍連続希釈液をMx3005でduplicateで解析した。QuantiTect Probe RT-PCR Kit を用いた1ステップRT-PCR(PCR効率:101%)。
2ステップRT-PCRのパフォーマンスと同等の1ステップRT-PCR - B
IL1R2(interleukin 1 receptor, type II)に特異的なプライマーおよび FAM標識プローブを用いて、白血球から調製したRNA(100 ng~1 pg)の10倍連続希釈液をMx3005でduplicateで解析した。QuantiTect Probe RT-PCR Kit を用いた1ステップRT-PCR(PCR効率:104%)。
特異的なプライマーアニーリング
バランスの取れたKCl および(NH4)2SO4 の組み合わせは、プライマーおよびプローブのPCRテンプレートへの特異的なアニーリングを促進する。K+ は二本鎖DNAのリン酸基に結合し、プライマーとプローブのアニーリングを安定化する。NH4+ は、ミスマッチな塩基対間の水素結合を不安定化する。
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