QuantiTect PCR Kits

qPCRとSYBR Green I を使用する2ステップqRT-PCRの場合、また、qPCRと2ステップqRT-PCRさらにマルチプレックスqPCRと配列特異的プローブを使用する2ステップマルチプレックスqRT-PCRの場合

S_1231_GEF_PCR0049
商業用のバルク製品、カスタマイズ製品、最適化された製品が必要ですか? QIAGENでは、ロジスティクスやコンプライアンスなどのサポートも行っています。QIAGEN Strategic Partnerships & OEMにお問い合わせください。

QuantiTect Probe PCR Kit (200)

Cat. No. / ID:   204343

200 x 50 µl反応の場合:3 x 1.7 ml 2x QuantiTect Probe PCR Master Mix、2 x 2 ml RNase-Free Water
Kit
QuantiTect PCR Kit
QuantiTect PCR +UNG Kit
Detection type
Probe
Multiplex Probe
SYBR Green
Reactions
200
1000
QuantiTect PCR Kitsは分子生物学的アプリケーション用であり、疾病の診断、予防、あるいは治療に使用することはできません。
商業用のバルク製品、カスタマイズ製品、最適化された製品が必要ですか? QIAGENでは、ロジスティクスやコンプライアンスなどのサポートも行っています。QIAGEN Strategic Partnerships & OEMにお問い合わせください。

特徴

  • 低コピーターゲットを高感度で検出
  • 配列特異的プローブまたはSYBR Greenを使用するqPCR
  • テンプレートの複数のログにわたる正確な定量
  • 反応条件やサイクリング条件の最適化が不要
  • 同じチューブで最大5標的を検出

製品詳細

QuantiTect PCR Kits(qPCRキット)は、real-time PCRと配列特異的プローブまたはSYBR Green I検出を使用する2ステップRT-PCRによって、gDNAおよびcDNA標的の高感度定量を可能にします。また、real-time PCRキットでは、1本のチューブで、マルチプレックス、real-time PCR、または2ステップRT-PCRによって、最大5つのgDNAまたはcDNA標的を確実に定量できます。すぐに使えるマスターミックスにホットスタートと独自のPCRバッファーシステムを組み合わせることで、最適化の必要なく、どのリアルタイムサイクラーでも高感度のqPCRが保証されます。dNTPミックスにはdUTPが含まれており、オプションでUNGによる処理が可能です。QuantiTect PCR Kitのマスターミックスは2–8°Cで保存でき、便利です。

配列特異的プローブを使用するマルチプレックスPCRには、蛍光正規化にROX色素を必要とするサイクラー用のQuantiTect Multiplex PCR Kitと、その他すべてのサイクラー用のQuantiTect Multiplex PCR NoROX Kitという2種類のキット形式があります。

パフォーマンス

QuantiTect定量PCRキットに含まれるHotStarTaq DNA Polymeraseは、他のポリメラーゼに比べ、最も厳密なホットスタートを提供することによって、PCR反応の特異性を高めます。

QuantiTect SYBR Green PCR Kitsは、広い直線範囲で特異的定量を可能にします(図「 広い直線範囲での特異的定量」参照)。QuantiTect Reverse Transcription KitおよびQuantiTect Primer Assaysと組み合わせて使用すると、QuantiTect SYBR Green PCR Kitsは高感度で信頼性の高い結果を提供します(図「 特異的で高感度な定量」参照)。

QuantiTect Probe PCR Kitsは、QuantiTect Reverse Transcription Kitと組み合わせると、高感度で信頼性の高い結果を提供します(図「 高い感度と効率、広いダイナミックレンジ」参照)。独自のPCRバッファー組成によって、QuantiTect Probe PCR Kitsは低コピーDNA標的の高感度定量だけでなく、広い直線範囲での正確な定量を実現します(図「 real-time PCRにおける広いダイナミックレンジ」参照)。

QuantiTect Multiplex PCR Master Mixは、マルチプレックス反応のPCR産物が、対応するシングル増幅反応のPCR産物と同じ効率と感度で増幅されることを保証します(表“トリプレックスPCRおよびシングルプレックスPCRと同等の閾値サイクル(CΤ)”と図“ 4-プレックスおよびシングルプレックスPCRと同等の結果”参照)。

 

トリプレックスPCRおよびシングルプレックスPCRと同等の閾値サイクル(CT)
  t(8;14)転座配列の検出(20コピー) GAPDH cDNA配列の検出(106コピー) NFKB cDNA配列の検出(コピー数については最初の列を参照)
105コピーのNFKBを用いるトリプレックスPCR 34.31 20.37 21.92
対応するシングルプレックスPCR 34.07 20.54 21.83
104コピーのNFKBを用いるトリプレックスPCR 34.61 20.62 25.03
対応するシングルプレックスPCR 34.00 20.46 25.19
103コピーのNFKBを用いるトリプレックスPCR 35.17 19.94 28.38
対応するシングルプレックスPCR 34.43 20.50 28.65

同じ反応中の参照遺伝子のコピー数が最大106倍多い場合でも、キットを用いれば、10コピー数程度の少数の標的遺伝子を検出できます(図「“ 過剰な参照遺伝子による10コピー標的遺伝子の検出」および対応する表「低存在量および高存在量の標的の定量的トリプレックスqPCRの成功」を参照)。

 

低存在量および高存在量の標的の定量的トリプレックスreal-time PCRの成功
  CSBGの検出 GAPDHの検出 HSPの検出
テンプレートミックス1 1400コピー 106コピー 2 x 104コピー
トリプレックスPCRのCT 27.73 18.69 23.59
シングルプレックスPCRのCT 27.08 18.89 23.52
テンプレートミックス2 140 コピー 106コピー 2 x 103コピー
トリプレックスPCRのCT 31.11 19.00 27.05
シングルプレックスPCRのCT 30.66 18.61 26.97
テンプレートミックス3 14コピー 106コピー 2 x 102コピー
トリプレックスPCRのCT 34.74 18.98 30.71
シングルプレックスPCRのCT 33.84 19.01 30.38
図参照

原理

QuantiTect SYBR Green PCR Kitsには、SYBR Green Iを使用してcDNA標的を高い特異性と感度でリアルタイム定量できるように最適化されたすぐに使用できるマスターミックスが含まれています(表“2x QuantiTect SYBR Green PCR Kitのコンポーネント”参照)。マスターミックスに含まれる蛍光色素SYBR Green Iは、数多くのさまざまな標的の分析を可能にし、標的特異的な標識プローブを合成する必要はありません。PCRバッファー中のK+イオンとNH4+イオンのバランスのとれた組み合わせは、特異的プライマーアニーリングを促進し、高いPCR特異性と感度を可能にします(図「特異的プライマーアニーリング」参照)。さらに、HotStarTaq DNA Polymeraseは厳密なホットスタートを提供し、非特異的産物の形成を防ぎます。

 

2x QuantiTect SYBR Green PCR Kitのコンポーネント
コンポーネント 特徴 メリット
HotStarTaq DNA Polymerase 95ºC 15分で活性化 室温でqPCR反応をセットアップ
QuantiTect SYBR Green PCR Buffer NH4+ イオンと K+ イオンのバランスの取れた組み合わせ 特異的なプライマーアニーリングにより信頼性の高いPCR結果を保証
dNTPミックス dTTPを部分的に置換し、オプションで反応のUNG処理を可能にするdUTPを含む オプションのUNG処理により、PCR産物のキャリーオーバーによるコンタミネーションを排除
SYBR Green I色素 二本鎖DNAと結合すると強い蛍光シグナルを発する 高感度定量
ROX色素 Applied BiosystemsおよびAgilentの装置(オプション)での蛍光シグナルの正規化用 ROX色素を必要とするサイクラーでの正確な定量。他のリアルタイムサイクラーの反応に干渉しない

QuantiTect Probe PCR Kitsには、配列特異的プローブを使用してgDNAおよびcDNA標的を高い特異性と感度でリアルタイム定量できるように最適化されたすぐに使用できるマスターミックスが含まれています(表“2x QuantiTect Probe PCR Kitのコンポーネント”参照)。このキットは、加水分解プローブ検出(たとえば、TaqMan®やその他の二重標識プローブ)、FRETプローブ、Molecular Beaconを含むあらゆるタイプの配列特異的プローブに使用できるように設計されています。QuantiTect Probe PCR Kitsは、K+イオンとNH4+イオンのバランスの取れた組み合わせを含む独自のPCRバッファーを含んでおり、特異的プライマーアニーリングを促進し、高いPCR特異性と感度を可能にします(図「特異的プライマーアニーリング」参照)。さらに、HotStarTaq DNA Polymeraseは厳密なホットスタートを提供し、非特異的産物の形成を防ぎます。

 

2x QuantiTect Probe PCR Kitのコンポーネント
コンポーネント 特徴 メリット
HotStarTaq DNA Polymerase 95ºC 15分で活性化 室温でqPCR反応をセットアップ
QuantiTect Probe PCR Buffer NH4+ イオンと K+ イオンのバランスの取れた組み合わせ 特異的なプライマーアニーリングにより信頼性の高いPCR結果を保証
dNTPミックス dTTPを部分的に置換し、オプションで反応のUNG処理を可能にするdUTPを含む オプションのUNG処理により、PCR産物のキャリーオーバーによるコンタミネーションを排除
ROX色素 Applied BiosystemsおよびAgilentの装置(オプション)での蛍光シグナルの正規化用 ROX色素を必要とするサイクラーでの正確な定量。他のリアルタイムサイクラーの反応に干渉しない

QuantiTect Multiplex PCR Kitsは、マルチプレックスの2ステップRT-PCRを1回目の試行で成功させることができます(フローチャート「 QIAGENマルチプレックスキット」参照)。最適化されたマスターミックスは、マルチプレックス反応のPCR産物が、対応する単一増幅反応のPCR産物と同じ効率と感度で増幅されることを保証します。このキットを使用すると、わずか10コピーという少数の標的遺伝子を検出できます。

参照遺伝子とターゲット遺伝子を別々の反応ではなく、同じ反応で増幅すると、取り扱いエラーが最小限に抑えられ、遺伝子定量の信頼性が向上します。QuantiTect Multiplex PCR Master Mixは、K+イオンとNH4+イオンのバランスの取れた組み合わせと独自の合成 Factor MPを含み、これらはともにプライマーとプローブの核酸テンプレートへの安定した効率的なアニーリングを促進し、高いPCR効率を可能にします(表「2x QuantiTect Multiplex PCR Kitのコンポーネント」参照)。さらに、HotStarTaq DNA Polymeraseは厳密なホットスタートを提供し、非特異的産物の形成を防ぎます。

QuantiTect PCR KitsのマスターミックスにはdUTPも含まれているので、PCR開始前にウラシル-N-グリコシラーゼ(UNG)で前処理することができるため、確実にPCR産物のコンタミネーションがその後のPCR反応に影響を与えないようにすることができます。

 

2x QuantiTect Multiplex PCR Kitのコンポーネント
コンポーネント 特徴 メリット
HotStarTaq DNA Polymerase 95ºC 15分で活性化 室温でqPCR反応をセットアップ
QuantiTect Multiplex PCR Buffer NH4+ イオンと K+ イオンのバランスの取れた組み合わせ 特異的なプライマーアニーリングにより信頼性の高いPCR結果を保証
合成Factor MP 同じチューブで最大4つの遺伝子を高い信頼性でマルチプレックス分析
dNTPミックス dTTPを部分的に置換し、オプションで反応のUNG処理を可能にするdUTPを含む オプションのUNG処理により、PCR産物のキャリーオーバーによるコンタミネーションを排除
ROX色素* Applied BiosystemsおよびAgilentの装置(オプション)で蛍光シグナルを正規化 ROX色素を必要とするサイクラーでの正確な定量。他のリアルタイムサイクラーの反応に干渉しない
図参照

操作手順

必要に応じて、反応をウラシル-N-グリコシラーゼ(UNG)で前処理し、以前の反応からのPCR産物のキャリーオーバーを除去できます。リアルタイム2ステップRT-PCRで最適な結果を得るには、QuantiTect Reverse Transcription Kitを使用してcDNAを合成することをお勧めします。このキットは、ゲノムDNAのコンタミネーションを総合的に除去しながら、わずか20分でcDNAを高速合成します。

QuantiTect SYBR Green and Probe PCR Kitsは、面倒で時間がかかる可能性がある反応条件の最適化を必要としません。すぐに使えるPCRマスターミックスにプライマーとDNAテンプレートを加え、反応を開始させるだけです。ハンドブックのプロトコールに従って、あらゆるリアルタイムサイクラーで迅速に信頼性の高い結果が得られます。

QuantiTect SYBR Green PCR KitsをQuantiTect Primer Assaysと組み合わせて使用すると、遺伝子発現分析で非常に特異性の高い結果が保証されます。これらは、ヒト、マウス、ラット、その他数多くの種の転写産物を検出するための、ゲノムワイドでバイオインフォマティクス的に検証されたプライマーセットです。QuantiTect Primer Assaysは、GeneGlobeで簡単にオンライン注文できます。

QuantiTect Multiplex PCR Kitsには、反応条件やサイクリング条件を最適化する必要のないすぐに使えるマスターミックスが含まれています。ハンドブックには、利用可能なすべてのリアルタイムサイクラーで使用できる単一プロトコールが含まれており、推奨色素もリストされています。

キットはマスターミックス中にROXパッシブレファレンス色素を含むものと含まないものとがあり、実質的にどのリアルタイムサイクラーでも使用できます(表“適切なQuantiTect Multiplex PCR Kitの選択”参照)。ROX濃度が最適化されているため、低いコピー数であっても自動データ分析により検出が可能です。

 

適切なQuantiTect Multiplex PCR Kitの選択
ROX色素 キット 互換性のあるサイクラー
マスターミックスに含まれる QuantiTect Multiplex PCR Kit Applied Biosystemsのサイクラー
マスターミックスに含まれない QuantiTect Multiplex PCR NoROX Kit Rotor-Geneサイクラー、およびBio-Rad、Cepheid、Eppendorf、Roche、Agilent、その他のサプライヤーのサイクラー

アプリケーション

QuantiTect PCR Kitsは、あらゆるリアルタイムサイクラーでcDNAの遺伝子発現分析やgDNAの定量に使用できます。これにはApplied Biosystems、Bio-Rad、Cepheid、Eppendorf、Roche、Agilentの装置が含まれます。Rotor-Gene Qやその他のRotor-Geneサイクラーには、これら装置での高速サイクリング用に特別に開発されたRotor-Gene SYBR Green PCR Kit、Rotor-Gene Probe PCR Kit、またはRotor-Gene Multiplex PCR Kitの使用をお勧めします。

 

QuantiTect PCR Kitsの比較
特徴 QuantiTect SYBR Green PCR Kits QuantiTect Probe PCR Kits QuantiTect Multiplex PCR Kits
アプリケーション ゲノムDNAまたはcDNA標的のリアルタイム定量 ゲノムDNAまたはcDNA標的のリアルタイム定量 ゲノムDNAまたはcDNA標的のリアルタイム定量
反応タイプ PCRと2ステップRT-PCR PCRと2ステップRT-PCR マルチプレックスPCRとマルチプレックス2ステップRT-PCR
リアルタイムまたはエンドポイント リアルタイム リアルタイム リアルタイム
サンプル/ターゲットタイプ DNA、cDNA DNA、cDNA DNA、cDNA
シングルプレックスまたはマルチプレックス シングル シングル マルチプレックス
SYBR Green Iまたは配列特異的プローブ SYBR Green I 配列特異的プローブ 配列特異的プローブ
サーマルサイクラー すべてのリアルタイムサイクラー(LightCycler, Rotor-Gene, ABIなど) ほとんどのリアルタイムサイクラー(LightCycler、Rotor-Gene、ABIなど) ほとんどのリアルタイムサイクラー(LightCycler、Rotor-Gene、ABIなど)
ROXあり/なし ROXあり ROXあり ROXあり/なし

裏付けデータと数値

リソース

クイックスタートプロトコール (4)
キットハンドブック (5)
QuantiTect Multiplex PCR Kit - with master mix containing ROX passive reference dye QuantiTect Multiplex PCR NoROX Kit - with master mix free of ROX passive reference dye For quantitative, multiplex, real-time PCR and two-step RT-PCR using sequence-specific probes  
For use with QuantiTect PCR Kits to eliminate carryover of PCR products
For quantitative, real-time PCR and two-step RT-PCR using sequence-specific probes
For genomewide, ready-to-use real-time RT-PCR assays using SYBR Green detection
For quantitative, real-time PCR and two-step RT-PCR using SYBR Green I
MSDS (2)
Download Safety Data Sheets for QIAGEN product components.
Download Safety Data Sheets for QIAGEN product components.
Safety Data Sheets (1)
Certificates of Analysis (1)

FAQ

Why does my PCR product show up later when comparing the QuantiTect SYBR Green PCR Kits with Roche kits using the same annealing temperature?
QuantiTect SYBR Green PCR Kits contain buffers with different salt concentrations compared to Roche Kits, resulting in different template melting temperatures. We recommend to decrease the annealing temperature by at least by 5°C when using a QuaniTect SYBR Green PCR Kit.
FAQ ID -1083
Can Uracil-N-Glycosylase (UNG) be added to the QuantiTect PCR Master Mix and be stored for later use?

So far, we have seen excellent stability of UNG in QuantiTect PCR Master Mixes. Therefore, UNG can be added to a fresh tube of master mix and be stored as recommended for the master mix in the respective handbook.

 

 

FAQ ID -2138
How do I setup and validate a multiplex PCR assay with QIAGEN PCR kits?

Ensure PCR amplicons are as short as possible, ideally 60–150 bp. Always use the same algorithm or software to design the primers and probes. For optimal results, only combine assays that have been designed using the same parameters.

 

Check the functionality of each set of primers and probes in individual assays before combining the different sets in the multiplex assay. Choose compatible reporters and quenchers based on a specific instrument. See How do I select appropriate reporter and quencher combinations for multiplex PCR.

 

FAQ ID -9093
I would like to order the QuantiFast Probe Assays without master mix. Is this possible?

QuantiFast Probe Assays are only sold in combination with optimized real-time RT-PCR master mixes and cannot be sold as stand-alone assays.

 

FAQ ID -2360
Does SYBR Green dye present in QuantiTect kits interfere with ethidium bromide staining?
SYBR Green dye does not interfere with ethidium bromide staining, and realtime SYBR Green PCR products can be visualized on an agarose gel.
FAQ ID -312
Do you have any information or guidelines regarding the choice of reference genes for real-time PCR?

Yes, please visit our website section 'Using endogenous control genes in real-time RT-PCR' for general information. It provides a list of relative gene expression levels for commonly used human and mouse reference genes.

We offer a set of ready-to-order control genes for use in SYBR Green based as well as probe based real-time RT-PCR.

In addition, you may want to refer to the following citations on reference gene selection for quantitative real-time PCR:

• Vandesompele J, De Preter K, Pattyn F, Poppe B, Van Roy N, DePaepe A, Speleman F [2002]: Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes. Genome Biol 2002, 3:0034.

• Radonic A, Thulke S, Mackay IM, Landt O, Siegert W, Nitsche A., 2004. Guideline to reference gene selection for quantitative real-time PCR. Biochem Biophys Res Commun. 313(4): 856-62.

• Katrien Smits,Karen Goossens, Ann Van Soom, Jan Govaere, Maarten Hoogewijs, Emilie Vanhaesebrouck,Cesare Galli, Silvia Colleoni, Jo Vandesompele, and Luc Peelman [2009]Selection of reference genes for quantitative real-time PCR in equine in vivo and fresh and frozen-thawed in vitro blastocysts. BMC Res Notes. Dec 11;2:246.

FAQ ID -2371
Do you recommend 1-step or 2-step real-time RT-PCR for gene expression analysis?

In one-step RT-PCR, both reverse transcription and amplification are performed in the same tube. Upon completion of reverse transcription, the reaction temperature is raised to reach denaturation/PCR enzyme activation temperature and the thermal cycling (PCR) begins. One-step RT-PCR generally uses gene-specific primers for both the RT and PCR steps. The procedure is fast, easy to automate, and minimizes the risk of contamination due to fewer handling steps.

In two-step RT-PCR, the RNA is first transcribed into cDNA using oligo-dT primers, random oligos, or gene-specific primers. An aliquot of the RT reaction is subsequently added to the real-time PCR reaction in a second tube. Choice of different types of RT primers allows the analysis of different transcripts by PCR from one RT reaction. Most commonly, an oligo-dT primer is used for the RT step, followed by PCR with a pair of gene-specific primers. Precious RNA samples can be immediately transcribed into more stable cDNA for later use and long-term storage.

 

The advantages of each method are summarized below:

Two-step RT-PCR One-step RT-PCR
Multiple PCRs from one RT reaction Easy handling
Flexibility with RT primer choice Fast procedure
Enables long-term storage of cDNA High reproducibility
  Low contamination risk

 

Optimized one-step and two-step RT-PCR kits compatible with any real-time cycler are available from QIAGEN. For further details, please see our online section on 'Critical factors for successful gene expression assays', or download our Brochure 'Critical Factors for Successful Real-Time PCR'.

FAQ ID -1056
Can I do 4-plex real-time PCR, on the ABI PRISM 7000, 7700, or 7900 using the QuantiTect Multiplex PCR Kit?
No. Due to hardware limitations, the maximum capacity of these real-time cyclers is triplex PCR.
FAQ ID -716
Will Uracil-N-Glycosylase (UNG) completely remove contaminating amplicons when using QuantiTect +UNG Kits?

This depends on the amount of contaminating amplicon. If the contamination is high (e.g., 10e6 copies), then there will still be traces of amplicon remaining. However, we expect a reduction by a factor of around 1000 using QuantiTect +UNG Kits (i.e., after UNG pretreatment, only 0.1% of the initial contamination will still be present in the reaction).

 

FAQ ID -2139
Why is the reaction volume for QuantiFast PCR Kits lower than that for QuantiTect PCR Kits?

The reduced reaction volume recommended for QuantiFast PCR Kits compared to QuantiTect PCR Kits allows more efficient temperature transfer during short cycling steps.

 

FAQ ID -1447
Why are the QuantiTect and QuantiFast Multiplex PCR Kits limited to triplex real-time PCR on some cyclers?

The presence of ROX as passive reference dye in the Master Mix of the QuantiTect Multiplex PCR Kit and the QuantiFast Mutliplex PCR Kit limits the use of these kits to triplex PCR on 4-channel real-time cyclers. This is because the ROX dye occupies the channel for detecting probes labeled with ROX, Texas Red, or other equivalent dyes.

However, 4-plex PCR is possible on on instruments equipped with at least 5 channels. Alternatively, if you have a 4-channel real-time cycler that does not use ROX passive reference dye (e.g., iCycler iQ, Rotor-Gene 3000, Mx4000, Mx3000P, Smart Cycler II, LightCycler 2.0), you can use the QuantiTect Multiplex PCR NoROX Kit or the QuantiFast Multiplex PCR +R Kit to perform 4-plex PCR.

Please see table “Real-Time, Multiplex PCR on a Wide Range of Real-Time Cyclers” for compabilities of the QuantiTect Multiplex kits with different real-time cyclers.

FAQ ID -715
Why do replicates in real-time PCR have different plateau heights?

Replicates in real-time PCR may have different plateau heights due to differences in the reaction kinetics for each sample. Even though replicates start out with identical template amounts, the rate at which reagents are being depleted, and the point when exponential accumulation of PCR product stops and becomes linear, differ between replicates. This will result in different plateau heights, the stage where PCR reactions have come to a halt, and little or no additional PCR product is being amplified. You can find further information in Chapter 'Quantification of target amounts' of our Brochure "Critical Factors for Successful Real-Time PCR".

 

FAQ ID -539
How should fluorescent labeled probes be stored?

Fluorescent oligonucleotides should be stored in the dark, as light can slowly degrade the fluorescent moieties. For optimal long-term storage of fluorescent dye-labeled probes (except Cyanine 570, Cy3.5, Cyanine 670, and Cy5.5), the oligos should be resuspended in a slightly basic solution (e.g., TE buffer at pH 8.0). If resuspended below pH 7.0, the probe can degrade. We recommend to aliquot the sample, and store the aliquots at -20°C.

Note that Cyanine 570, Cy3.5, Cyanine 670, and Cy5.5 begin to degrade at a pH above pH 7.0. For best results, resuspend Cy-labeled oligos at pH 7.0, aliquot, lyophilize, and store at -20°C.

FAQ ID -784
Why is an ice-incubation step included during reaction set-up when following the QuantiTect RT-PCR but not the QuantiTect PCR protocol.
The ice step is recommended to prevent non-specific reverse transcription during reaction set-up. Mispriming by Taq is not a concern in these cases because both QuantiTect RT-PCR and PCR kits contain HotStarTaq, a DNA polymerase that only becomes active following a 15 minute, 95 degree incubation step.
FAQ ID -283
Do the master mixes in Rotor-Gene Kits contain dUTP to allow UNG pretreatment?

No. The master mixes in Rotor-Gene Kits contain dTTP instead of dUTP. If UNG treatment is required, we recommend using QuantiTect +UNG Kits. QuantiTect Kits are also compatible with the Rotor-Gene Q; however, the kits require a significantly longer cycling time.

 

 

FAQ ID -2117
If template abundance differs significantly between housekeeping and target gene, are both amplified equally efficient with the QuantiTect Multiplex Kits?
Yes. The pre-optimized master mix ensures that all gene targets in a multiplex reaction are amplified with the same efficiency and sensitivity as in corresponding "single-plex" reactions, independent of starting copy numbers.
FAQ ID -718
How do I create a workspace that is free of DNA contamination, prior to carrying out a qPCR experiment?

Any DNA contamination will artificially inflate the SYBR Green signal, yielding skewed gene expression profiles and false-positive signals. The most common source of DNA contamination is from PCR products generated during previous experiments. Such contamination is most often due to the improper disposal of tubes, tips, and gels that previously came into contact with PCR products. Additionally, PCR products may also contaminate pipettors, racks, work pads, and commonly used reagents such as water and buffers. To minimize the risk of contaminating your experiment with extraneous DNA, the following steps should be taken:

 

  • Remove a single aliquot of water from your PCR-grade stock, sufficient to complete the experiment. This minimizes the number of times that the stock container is opened, thereby minimizing contamination risks.
  • Use only fresh PCR-grade reagents and disposable labware.
  • Treat any labware (tubes, tips, and tip boxes) used in PCR with 10% bleach, before discarding.
  • Maintain a dedicated workspace for PCR setup (perhaps a PCR-only hood), away from areas of the lab where post-PCR work is done, such as running gels, enzyme digestions, and cloning.
  • Change the lab bench pads/papers often and decontaminate lab benches and labware (racks, pipettors, etc.) before each use by washing with 10% bleach, and/or exposing to UV light for at least 10 minutes. This serves to degrade and/or inactivate contaminating DNA.
  • Before, during, and after the experiment, minimize the opening and closing of any tubes or plates used during the experiment.  
FAQ ID -2654
How long should the amplicon be if I am using QuantiTect SYBR Green detection?
For accurate quantification we recommend an amplicon length of 100-150 bp to ensure optimal amplification efficiency.
FAQ ID -553
What reaction volume should I use with QuantiTect Kits?
QuantiTect Kits for real-time PCR and RT-PCR have been optimized for a final reaction volume of 50 µl for use with single tubes and 96-well plates, or 20 µl for use with 384-well plates and LightCycler capillaries. We strongly recommend using the primer and probe concentrations, reaction volumes, and amount of starting template given in the QuantiTect handbooks. However, volumes may be reduced to 20 µl and 10 µl, respectively.
FAQ ID -551
Do you have information on the use of recombinant DNA and RNA as absolute standards for realtime RT-PCR?

Recombinant DNA (recDNA) is very stable and represents the average size of mRNA. Due to the cloning and purification processes, obtaining recDNA can lengthen the overall process of generating standards.

Recombinant RNA (recRNA) and native RNA undergo reverse transcription as well as PCR, and mimic the natural process for mRNA in RT-PCR. Complicated cloning and purification of recRNA and instability of recRNA are two disadvantages for using recRNA as a standard. For further details please refer to the section "Generating Standard Curves" in Appendix D of the QuantiTect SYBR Green PCR Handbook.

FAQ ID -729
What is a QuantiTect Primer Assay?

QuantiTect Primer Assays are primer pairs designed and bioinformatically validated specifically for real-time RT-PCR with SYBR Green detection. To find a primer assay for your target gene of interest, please visit our GeneGlobe data base.

For best results, we strongly recommend using QuantiTect Primer Assays in combination with QIAGEN's products for SYBR Green-based Real-Time PCR and RT-PCR.

FAQ ID -1141
What is the concentration of the primers in a reaction using the QuantiTect Primer Assays?

The primers of the QuantiTect Primer Assays are at a proprietary concentration that was specially optimized for sensitive and efficient amplification in any real time cycler. Always dilute these primers to a final work solution of 1x in your reaction, using either the QuantiTect SYBR Green PCR Kit or the QuantiTect SYBR Green RT-PCR Kit. Follow the instructions for use of the Primer Assays in the QuantiTect Primer Assay handbook.

FAQ ID -850
Can I use uracil-N-glycosylase (UNG) with the QuantiFast and Rotor-Gene PCR kits?

No. UNG treatment does not provide any advantage for the QuantiFast and Rotor-Gene PCR kits because the mastermixes do not contain dUTP. Use the QuantiTect kits if you intend to use the UNG treatment.

FAQ ID -9092
My QuantiTect Master Mix did not freeze at degrees. Is it still ok to use?
Yes. The QuantiTect Master Mix may not necessarily freeze at -20 degrees. The performance is not affected as long as the correct storage temperature is maintained.
-20
What is a quenched FRET assay?
Quenched FRET assays are similar to FRET assays except that the decrease in energy of the donor fluorophore is measured instead of the increase in energy of the acceptor fluorophore.
FAQ ID -542
How many template copies can I detect using QuantiTect Kits in real-time PCR?
The number of template copies that can be detected during real-time PCR depends on template and primer quality, as well as on the amplification reagents used. As few as 10 copies can typically be detected with the QuantiTect Probe PCR and QuantiTect SYBR Green PCR Kits.
FAQ ID -543
What annealing temperature should be used with the QuantiTect Primer Assays?

The annealing temperature for QuantiTect Primer Assays should be 55oC when used with the QuantiTect SYBR Green PCR Kit and the QuantiTect SYBR Green RT-PCR Kit.

Use of the QuantiFast SYBR Green PCR and QuantiFast SYBR Green RT-PCR Kits requires a combined annealing/extension step at 60oC, as described in the QuantiTect Primer Assay Handbook.

Note that these Assays are guaranteed for use with the QuantiTect or QuantiFast chemistries only!

 

 

FAQ ID -849
Why is the storage time for QuantiFast PCR Kits shorter than that for QuantiTect PCR Kits?

The storage time for QuantiFast PCR Kits is shorter than for QuantiTect PCR Kits, because all QuantiFast master mixes contain HotStarTaq Plus DNA Polymerase, instead of HotStarTaq DNA Polymerase which requires longer activation times.

Excessive exposure to elevated temperatures will result in reactivation of the HotStarTaq Plus DNA Polymerase, eventually leading to nonspecific amplification.

 

FAQ ID -1446
Why do I see multiple high-intensity peaks in my qPCR dissociation curve at temperatures less than 70ºC?

If the extra peaks seem irregular or noisy, do not occur in all samples, and occur at temperatures less than 70 ºC, then these peaks may not represent real PCR products and instead may represent artifacts caused by instrument settings.

 

Usually extra peaks caused by secondary products are smooth and regular, occur reproducibly in most samples, and occur at temperatures greater than 70 ºC. Characterization of the product by agarose gel electrophoresis is the best way to distinguish between these cases. If only one band appears by agarose gel then the extra peaks in the dissociation curve are instrument artifacts and not real products. If this is the case, refer to the thermal cycler user manual, and confirm that all instrument settings (smooth factor, etc.) are set to their optimal values.

 

FAQ ID -90990
How do I quantify gene expression levels if the amplification efficiencies are different between the genes of interest and endogenous reference gene?

The REST 2009 (Relative Expression Software Tool) software applies mathematic models that compensate for the different PCR efficiencies of the gene of interest and reference genes. In addition, the software can use multiple reference gene normalization to improve the reliability of result, as well as provides statistical information suitable for robust comparison of expression in groups of treated and untreated. QIAGEN offers the REST 2009 software free of charge.

FAQ ID -9095
Have QuantiTect Primer Assays been tested with QuantiFast SYBR Green PCR Kits on the Mastercycler ep realplex?

Yes, QuantiTect Primer Assays work very well under fast-cycling conditions. Results achieved with QuantiFast SYBR Green Kits are comparable to those achieved with QuantiTect SYBR Green Kits.

 

FAQ ID -1714
Can Uracil-N-Glycosylase (UNG) be purchased separately, independent of QuantiTect PCR Kits?
No, UNG is only available in combination with a QuantiTect PCR Kit.
FAQ ID -2134
What is the threshold cycle or Ct value?
The Ct or threshold cycle value is the cycle number at which the fluorescence generated within a reaction crosses the fluorescence threshold, a fluorescent signal significantly above the background fluorescence. At the threshold cycle, a detectable amount of amplicon product has been generated during the early exponential phase of the reaction. The threshold cycle is inversely proportional to the original relative expression level of the gene of interest.
FAQ ID -2682
Why are my realtime PCR amplification plots hook-shaped?
During the late phase of PCR, as a lot of PCR product has been generated, there is strong competition between hybridization of the probe to the target strand and re-association of the two complementary product strands. For some primer–probe combinations, re-association occurs more quickly than probe hybridization towards the end of the PCR. Therefore, the yield of PCR product seems to decrease after reaching a peak. For accurate quantification, fluorescence data is measured during the log-linear phase of the reaction, i.e., during the initial increase of fluorescence, before this phenomenon occurs.
FAQ ID -587
QuantiTect Primer Assays are bioinformatically validated, genomewide primer sets. What does “bioinformatically validated” mean?

For each QuantiTect Primer Assay, we retrieve the sequence of the target gene from curated databases and exclude SNP regions from assay design.

We then use our proprietary algorithm to design assays that amplify RNA sequences only (i.e., at least one primer overlaps a splice site), provided that information on the position of splice sites is available. The assays are designed to provide optimal performance with QuantiFast and QuantiTect SYBR Green Kits.

After assay design, we validate randomly selected QuantiTect Primer Assays using real-time RT-PCR to check their compatibility with various real-time cyclers. Both two-step and one-step RT-PCR are carried out. Successful amplification of the target is indicated by the following factors: high sensitivity, high efficiency, high specificity (i.e., a single peak in melting curve analysis), and no primer–dimers in the no-template control (NTC).

To find a QuantiTect Primer Assay for your target gene of interest, please visit our GeneGlobe data base.

 

FAQ ID -1982
Do I need to determine limiting primer concentrations with the QuantiTect Multiplex PCR Kits?
No, that is not necessary. Simply use the primer concentrations specified in the protocols in the supplied QuantiTect Multiplex PCR handbooks.
FAQ ID -714
What do I do if no fluorescent signal is detected in a real-time PCR assay?

Check the template quality and integrity by amplifying an endogenous control gene. Check the amplicon by QIAxcel Advanced system or agarose gel electrophoresis to show that amplification was successful.

 

Determine whether the gene of interest is expressed in your sample. See How can I find out if my gene of interest is express in a specific tissue type or cell line.  Ensure the assay setup and cycling conditions are correct, and that the data collection channel matches the emission wavelength of the fluorescent dye used. Use a control sample in which the gene of interest is definitely expressed.

 

If the issue persists, please send the original run file to QIAGEN Technical Services.

FAQ ID -9091
Can I increase the annealing temperature recommended for QuantiTect Primer Assays used with the QuantiTect SYBR Green PCR Kits?

QuantiTect Primer Assays are optimized for an annealing temperature of 55ºC when using the QuantiTect SYBR Green PCR Kit (for two-step RT-PCR) or the QuantiTect SYBR Green RT-PCR Kit (for one-step RT-PCR) .

Increasing the annealing temperature will reduce PCR performance (sensitivity and PCR efficiency) when using QuantiTect Primer Assays with these kits.

 

FAQ ID -1146
Do you have a protocol for performing quantitative multiplex PCR on the LightCycler 2.0 system?
How do I select appropriate reporter and quencher combinations for multiplex PCR?

For duplex analysis, using non-fluorescent quenchers (e.g., Black Hole Quencher®) is preferred over fluorescent quenchers (e.g., TAMRA fluorescent dye). For triplex and 4-plex analysis, QIAGEN strongly recommends using non-fluorescent quenchers. Generally, use the green channel, the yellow channel, and the orange and crimson channels to detect the least abundant target, the second least abundant target, and the two most abundant targets, respectively. For instrument-specific recommendations, please see the handbooks for the QuantiTect Multiplex PCR kit, QuantiFast Multiplex kit or Rotor-Gene Multiplex kit.

 

FAQ ID -9094
Do you have a protocol for adding new reporter dyes to the ABI PRISM 7900 Sequence Detection System?
How can I predict the percent qPCR signal due to contaminating DNA, for a given qPCR assay, and its matching NRT control?

Assuming 100% amplification efficiency, each step increase in Ct value represents a doubling in the amount of qPCR template. Therefore, evaluating the difference in Ct values between the qPCR assay, and its matching NRT control, leads to the following predictions:

CtNRT - Ct+RT Fraction of gene expression signal due to contaminating DNA Percentage of gene expression signal due to contaminating DNA
1 (1/21) = 1/2 50%
2 (1/22) = 1/4 25%
3 (1/23) = 1/8 13%
4 (1/24) = 1/16 6%
5 (1/25) = 1/32 3%

FAQ ID -2688
Can I skip the gDNA wipeout buffer treatment step for the QuantiTect Reverse Transcription Kit?

The gDNA wipeout buffer incubation step can be skipped when the total RNA is free from genomic DNA. However, the gDNA wipeout buffer is still required to be added because the reverse transcription step is optimized in the presence of components in the gDNA wipeout buffer.

FAQ ID -9098
Why does my realtime PCR assay quality decrease over time?
Make sure that template, primers, probes, and amplification reagents are stored correctly and avoid multiple freeze–thaw cycles for oligonucleotides and template. Check the performance of your real-time instrument as some instruments require the halogen lamp to be frequently replaced. Lasers must also be replaced occasionally.
FAQ ID -589
How can I tell if I have primer-dimers in my PCR reaction?
In quantitative (real-time) PCR, primer-dimers will appear as a peak with a Tm lower than the Tm of the specific product. This peak will also appear in the no-template control (NTC). In non-quantitative endpoint PCR, primer dimer will appear as a more or less faint smear on an agarose gel, below the product band of interest.
FAQ ID -552
What is a dissociation curve, and why is it important to run a dissociation curve, following qPCR using SYBR Green chemistry?

Dissociation curves are carried out at the end of a PCR experiment by following a 3-step procedure.

First, all the components are denatured at 95°C, followed by complete annealing at a set temperature (based on the primer Tm values), followed by a gradual increase in temperature up to 95°C. Fluorescence intensity is monitored during this final temperature increase, resulting in the generation of a melting curve or dissociation curve.

By analyzing the first derivative of such a curve, you can readily assess the homogeneity of the PCR products, including the presence of primer–dimers, thereby determining the specificity of the PCR reaction. It is important to carry out such post-PCR analyses when using SYBR Green probe chemistry due to this reagent's lack of sequence specificity.

FAQ ID -2678
Which real-time PCR kits are recommended downstream of the QuantiTect Whole Transcriptome Kit?

We highly recommend any QuantiTect or QuantiFast Kit for quantitative PCR on cDNA generated with the QuantiTect Whole Transcriptome Kit.

 

FAQ ID -1592
How much time will be saved when switching from standard cycling to fast cycling with QuantiFast Kits?

Depending on the qPCR instrument, time savings when switching from standard cycling (e.g., QuantiTect PCR Kits) to fast cycling using QuantiFast Kits range from 40% to 60%.

 

 

FAQ ID -1438
How much template can I use in the reaction and what is the maximum volume of template that can be used in the QuantiTect Kits?

Even when detecting low-abundance targets, we recommend using no more than 500 ng/reaction of DNA (for PCR kits) or RNA (for RT-PCR kits). Generally, 1–100 ng template should be sufficient and for abundant transcripts as little as 1 pg can be used.

Template purity is important if large volumes of low concentration template are to be added to the reaction. Using DNA or RNA purified with QIAGEN products, the template can contribute up to 40% of the final reaction volume as long as the recommended template amounts are not exceeded. If cDNA from an RT reaction is used as template, the volume of undiluted RT reaction added should not exceed 10% of the final PCR volume.

FAQ ID -1086
How should I handle and store absolute quantitation standards for real-time experiments?
Store the standards at a high concentration in aliquots at -20oC to -70oC. If using low concentrations, stabilize standards with carrier nucleic acid. It is always best to use freshly diluted standards for each experiment. If possible, use siliconized tubes for standard (and target) dilutions. This will prevent any unspecific binding of nucleic acids to the plastic.
FAQ ID -9099
How can one perform multiplex real-time PCR analysis using the LightCycler 2.0?
The LightCycler 2.0 requires a color compensation file to separate the fluorescent signals and eliminate crosstalk between the individual detection channels. Please contact QIAGEN Technical Services for a specialized protocol.
FAQ ID -780
How do I ensure reliable results for High Resolution Melting (HRM) assays?

Reliable HRM analysis results depend on template quality, highly specific HRM PCR kit with a saturation dye, a real-time instrument with HRM capability, and powerful software package. Factors critical for successful HRM analysis are:

 

  • Use the same genomic DNA purification procedure for all samples being analyzed by HRM. This avoids variation due to differing composition of elution buffers.
  • DNA template concentrations should be normalized using the same dilution buffer. Ensure the CT values are below 30 and differ no more than 3 CT values across individual samples.
  • Design assays with amplicon length 70–350 bp. For SNP analysis, use amplicon length 70–150 bp.
  • Always start with 0.7 µM primer concentration

 

For more details, please refer to the HRM Technology – FAQs and the Critical Success Factor for HRM performance.

FAQ ID -9097
Do QuantiTect Primer Assays contain SYBR Green dye?

No, QuantiTect Primer Assays are supplied as lyophilized, premixed primer pairs. Reaction components for SYBR Green real-time RT-PCR must be purchased separately.

To find a QuantiTect Primer Assay for your target gene of interest, please visit our GeneGlobe data base.

FAQ ID -1143
Where can I find info on compatible reporter dyes for use in real-time multiplex PCR using the QuantiTect Multiplex PCR Kits?

Compatible reporter dye combinations for different real-time cyclers are listed in the "Important Notes" section under 'Suitable combinations of reporter dyes' in the QuantiTect Multiplex PCR Handbook and the QuantiTect Multiplex PCR NoROX Handbook

 

 

 

FAQ ID -719
Do the QuantiTect SYBR Green Kits contain a stabilization reagent that inhibits the sensitivity of real-time PCR?
No. In fact, QuantiTect SYBR Green PCR Kits contain a stabilization reagent that increases the sensitivity of real-time PCR. The QuantiTect SYBR Green Kits also contain a carefully controlled concentration of SYBR Green that is sufficient to detect PCR fragments while having no inhibitory effect on the enzymatic reaction.
FAQ ID -328
Why should DNA or cDNA targets be less than 250 bp long for real-time PCR?

Shorter amplification products facilitate high PCR efficiencies. Ideally, amplicon length should be less than 150 bp for optimal amplification efficiency. PCR efficiencies close to 100% are a crucial prerequisite for accurate quantification of target copy numbers in real-time PCR.

FAQ ID-751
What should I use as a standard for absolute quantification in real-time PCR?

For quantification of RNA, we strongly recommend using RNA molecules as standards. Use of in vitro transcripts as standards takes into account the variable efficiency of the RT reaction. An alternative to the use of in vitro transcripts as RNA standards is the use of a defined RNA preparation (e.g., from a cell line or virus preparation), for which the absolute concentration of the target has already been determined.

For quantification of DNA, several types of DNA can be used, such as plasmids, PCR products, or genomic DNA.

For more information, see Appendix E 'Generating Standard Curves' in the QuantiTect Probe PCR Handbook.

FAQ ID -1085
Are QuantiTect PCR +UNG Kits available in bulk format (e.g., 25 ml master mix)?

QuantiTect PCR +UNG Kits are available as 200-reaction kits only.

 

FAQ ID -2136
What are the main differences between Rotor-Gene and QuantiTect or QuantiFast PCR Kits?

Rotor-Gene Kits are specifically developed for the Rotor-Gene Q PCR Cycler. The unique rotary system of the cycler combined with the kits’ proprietary buffer system enable ultrafast cycling. Rotor-Gene Kits do not contain ROX dye since no normalization to a passive reference is required. Also, Rotor-Gene Kits do not contain dUTP; therefore, UNG pretreatment is not possible.

 

FAQ ID -2119
Can I make a master mix with QuantiProbes, primers, and all other reaction components and store it for later use?

Yes, probes and primers can be added to the QuantiTect Probe PCR- and the QuantiTect Probe RT-PCR Master Mix. This master mix can be stored at -20°C for up to 50 days, with a maximum of six freeze-thaw cycles (tested in our labs).

The master mix must be stored protected from light to avoid bleaching of the fluorescent probe. However, the QuantiTect RT Mix should be stored separately at –20°C, because the reverse transcriptases are sensitive to freezing in an aqueous environment. The QuantiTect RT Mix should be added to this master mix just prior to adding the template RNA and placing the reactions into the thermal cycler.

FAQ ID -584
How do QuantiFast PCR Kits compare to QuantiTect PCR Kits for quantitative real-time PCR?

We have compared QuantiFast Kits and QuantiTect Kits using around 30 different assays (using both SYBR Green and Probe detection for each assay).

QuantiFast Kits gave identical or sometimes better Ct values than QuantiTect Kits (except for very long amplicons). Therefore, scientists switching from QuantiTect to QuantiFast Kits can, in most cases, obtain comparable results.

 

 

FAQ ID -1441
Is it possible to perform a UNG treatment when using QuantiTect kits?

Yes - dTTP in the QuantiTect Master Mix in QuantiTect SYBR Green and Probe PCR Kits is partially substituted by dUTP, therefore allowing treatment with uracil-N-glycosylase.

However, only heat-labile UNG can be used for one-step RT-PCR with the QuantiTect Probe RT-PCR and SYBR Green RT-PCR Kits. This is because heat stable UNG (isolated from E. coli) will destroy any cDNA synthesized during the RT step at 50°C. Heat-labile UNG will have lost any activity after a few minutes of the RT-step and therefore cannot interfere with cDNA synthesis.

For heat-stable uracil-N-glycosylase, please use the QuantiTect SYBR Green PCR +UNG (cat. no. 204163) or QuantiTect Probe PCR +UNG Kits (cat. no. 204363). Both kits include heat-stabile uracil-N-glycosylase. Although QIAGEN UNG is inactivated by incubation at 95°C for 15 minutes, the enzyme may exhibit residual activity at lower temperatures due to refolding. Therefore, it is recommended to perform subsequent PCR using a temperature of 55°C or above for the annealing step.

FAQ ID -564
Can I use the QuantiTect Multiplex PCR Kits on the Roche LightCycler systems with TaqMan® probes or QuantiTect Assays?
  • LightCycler: We do not recommend performing real-time, multiplex PCR using TaqMan® probes or QuantiTect Assays on the LightCycler system due to the limitations of its optical detection system.
  • LightCycler 2.0: Information on using the QuantiTect Multiplex PCR NoROX Kit with the LightCycler 2.0 system is provided in the QuantiTect Multiplex PCR NoROX Handbook. You will find the recommendation for the LightCycler 2.0 system in the chapter 'Important Notes' under "Suitable combinations of reporter dyes".

See trademarks.

FAQ ID -717
How do I avoid collecting a fluorescence reading from primer-dimer with the QuantiTect SYBR Green PCR Kit?

Depending on primer design and copy number of target, primer-dimer may occur and its signal might be detected. Typical strategies against this are to optimize PCR conditions and/or redesign the assay.

 

Alternatively, an additional data-acquisition step can be added to the 3-step cycling protocol. First, determine the melting temperatures (Tm) for both the amplicon and the primer-dimer. Then, add a 15 second data-acquisition step with a temperature that is higher than the primer-dimer Tm, but approximately 3ºC lower than the specific amplicon Tm.

FAQ ID -9096
What is the delta Rn value?
The Rn value, or normalized reporter value, is the fluorescent signal from SYBR Green normalized to (divided by) the signal of the passive reference dye for a given reaction. The delta Rn value is the Rn value of an experimental reaction minus the Rn value of the baseline signal generated by the instrument. This parameter reliably calculates the magnitude of the specific signal generated from a given set of PCR conditions. For more information, please refer to your cycler's user manual.
FAQ ID -2681
Do I need to calibrate my real-time cycler if I want to use Yakima Yellow?
The emission maximum of Yakima Yellow (552 nm) is almost identical to that of the fluorescent dye VIC. Therefore, the channel and filter settings for VIC can also be used for Yakima Yellow.
FAQ ID -540
Why do melting temperatures differ between PCR fragments amplified with QIAGEN's QuantiTect SYBR Green PCR Kits and Roche Kits?
The melting temperature of a PCR product depends on the salt concentration of the buffers used for amplification. Buffer compositions differ between QuantiTect SYBR Green PCR Kits and Roche kits, resulting in different PCR product melting temperatures.
FAQ ID -1084
Why do I see multiple high-intensity peaks in my qPCR dissociation curve at temperatures less than 70 ºC?

If the extra peaks seem irregular or noisy, do not occur in all samples, and occur at temperatures less than 70 ºC, then these peaks may not represent real PCR products and instead may represent artifacts caused by instrument settings.

Usually extra peaks caused by secondary products are smooth and regular, occur reproducibly in most samples, and occur at temperatures greater than 70 ºC. Characterization of the product by agarose gel electrophoresis is the best way to distinguish between these cases. If only one band appears by agarose gel then the extra peaks in the dissociation curve are instrument artifacts and not real products. If this is the case, refer to the thermal cycler user manual, and confirm that all instrument settings (smooth factor, etc.) are set to their optimal values.

 

 

FAQ ID -2690