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.

製品名 Cat. no. List price:
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
204443
¥83,500
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
204445
¥341,000

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。


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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.|
Performance

The QuantiTect Probe RT-PCR Kit has a unique RT-PCR buffer that promotes highly specific annealing of primers and probes to the PCR template. It contains HotStarTaq DNA Polymerase, which provides the most stringent hot start compared with other polymerases (see figure "Highly specific amplification").

HotStarTaq DNA Polymerase and the unique composition of the RT-PCR buffer enable the QuantiTect Probe RT-PCR Kit to provide sensitive quantification of low-copy RNA targets, as well as accurate quantification over a wide linear range (see figure "High sensitivity and efficiency, and wide dynamic range"). Performing reverse transcription and PCR sequentially in the same tube does not impair sensitivity, as demonstrated by CT values that are comparable to those achieved in real-time two-step RT-PCR (see figures "One-step RT-PCR with comparable performance to two-step RT-PCR — A" and "One-step RT-PCR with comparable performance to two-step RT-PCR — B", and table).

One-step RT-PCR with comparable performance to two-step RT-PCR.
Amount of cDNA/RNAMean CT value Mean CT value
(two-step RT-PCR) (one-step RT-PCR)
100 ng 24.66 24.66
10 ng 28.15 27.84
1 ng 31.42 31.49
0.1 ng 34.80 34.56
0.01 ng 37.78 37.66
CT values from the 2 amplification plots shown in figures "One-step RT-PCR with comparable performance to two-step RT-PCR — A" and "One-step RT-PCR with comparable performance to two-step RT-PCR — B".
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.

Components of 2x QuantiTect Probe RT-PCR Kit
Component Features Benefits
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.

Feature
Specifications
Applications Real-time quantification of RNA targets
Reaction type One-step RT-PCR
Real-time or endpoint Real-time
Sample/target type RNA targets
Single or multiplex Single
SYBR Green I or sequence-specific probes Sequence-specific probes
Thermal cycler All real-time cyclers (e.g. LC, RG, ABI)
With or without ROX With ROX

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キットハンドブック
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For quantitative, real-time one-step RT-PCR using sequence-specific probes
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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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MSDS
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Download Safety Data Sheets for QIAGEN product components.
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PCR and two-step RT-PCR.
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)。

Highly specific amplification.
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。
Wide dynamic range in one-step RT-PCR
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。

One-Step RT-PCR with Comparable Performance to Two-Step RT-PCR
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%)。

One-step RT-PCR with comparable performance to a two-step RT-PCR
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%)。

Specific primer annealing using a balanced combination of K+ and NH4+ ions
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。