【RT-qPCR原理與產品介紹】RT-qPCR定量逆轉錄聚合酶鏈反應-BIO-HELIX

RT-qPCR定量逆轉錄聚合酶鏈反應(Quantitative reverse transcription polymerase chain reaction)

【RT-qPCR原理與產品介紹】

什麼是 RT-qPCR？

定量逆轉錄聚合酶鏈反應(Quantitative reverse transcription polymerase chain reaction)，也稱為 RT-qPCR，用於檢測和量化 RNA。總 RNA (Total RNA )或 mRNA 首先被轉錄( transcription)成互補 DNA (complementary DNA, cDNA)。然後將 cDNA (complementary DNA, cDNA)用作定量 PCR (quantitative PCR)或實時 PCR 反應 ( real-time PCR reaction , qPCR) 的模板( template )。在 qPCR 中，在每個 PCR 循環中使用螢光測量擴增產物的量。RT-qPCR 用於多種應用，包括基因表達分析(gene expression analysis)、RNAi 驗證(RNAi validation)、微陣列驗證(microarray validation)、病原體檢測(pathogen detection)、基因檢測(genetic testing)和疾病研究。

一步驟與兩步驟 RT-qPCR

RT-qPCR 可以通過一步驟或兩步驟進行。一步驟分析將逆轉錄( reverse transcription)和 PCR 結合在一個試管和緩衝液中，使用逆轉錄酶(reverse transcriptase)和 DNA 聚合酶(DNA polymerase)。一步驟 RT-qPCR 僅使用序列特異性引物。在兩步分析中，逆轉錄和 PCR 步驟在不同的試管中進行，具有不同的優化緩衝液、反應條件和引發策略。一步驟RT-qPCR優點為由於兩個反應都發生在同一個試管中，因此實驗差異較小、適用於高通量擴增/篩選。一步驟RT-qPCR優點: 生成穩定的 cDNA 庫，可長期保存並用於多次反應

Bio-Helix在不斷發展，提供高品質 PCR、qPCR、RT-qPCR 和 RT-PCR，專為研究或要求苛刻的診斷產品應用而設計。Bio-Helix RScript 逆轉錄酶 (Bio-Helix RScript Reverse Transcriptase) – 專為研究和診斷應用而創新設計，滿足所有的 cDNA 合成需求，並克服寬溫度範圍內最具挑戰性的二級 RNA 結構( secondary RNA structures)。新一代工程重組 M-MLV 逆轉錄酶( M-MLV reverse transcriptase)具有更高的熱穩定性(thermostability)、持續合成能力(processivity)、穩健性(robustness)、最佳 cDNA 產量、用於降低 RNase H 活性的專有位點突變以及延長的半衰期，是世界上最通用的逆轉錄酶( versatile reverse transcriptase )不僅簡單地滿足常規 cDNA 合成要求，而且即使是手頭最具挑戰性的 RNA 樣品也能實現卓越的性能。RScript cDNA 合成試劑盒(RScript cDNA Synthesis Kit, RK001-0050) 專門用於研究和診斷應用，可滿足所有的 cDNA 合成需求；克服寬溫度範圍內最具挑戰性的二級 RNA 結構(secondary RNA structures)。套組內包含新一代工程重組 M-MLV 逆轉錄酶(M-MLV reverse transcriptase)，具有改進的熱穩定性( improved thermostability)、持續合成能力(robustness)、穩健性、最佳 cDNA 產量( optimal cDNA yields)、Moloney 小鼠白血病病毒逆轉錄酶 (M-MLV RT) 缺乏 DNA 核酸內切酶活性，具有較低的 RNase H 活性。是最通用的世界上的逆轉錄酶，不僅可以簡單地滿足常規 cDNA 合成要求，而且即使對於手頭最具挑戰性的 RNA 樣品也能實現卓越的性能。PanProbes™ 一步驟 RT-qPCR 試劑盒由於其 RScript 逆轉錄酶(RScript reverse transcriptase)、降低的 RNase H+ 活性 MMLV 酶以及強大的 RNase 抑制劑混合物(RNase inhibitors mix )而提供目標 RNA 水平的高靈敏度，旨在減少反應設置期間的 RNA 降解(RNA degradation)、錯誤引物(mispriming)、反轉錄(reverse transcription )以保證最佳的 RT 效率。Universal qPCR Master Mix 是一種 2x 濃縮的即用型預混液，針對基於探針的real-time PCR 進行了優化，並與大多數商用實時 real-time PCR systems (ROX-independent and ROX-dependent)兼容。它包含抗體介導的熱啟動 Taq DNA 聚合酶( antibody-mediated hot-start Taq DNA polymerase)、dNTP、MgCl2、增強劑(enhancers)、穩定劑(stabilizers)和成功進行 PCR 反應( success PCR reaction)的必需品。另提供即用型 qPCR 混合液、PanProbes™ Universal qPCR預混液、RNase 抑制劑、一個步驟RT-qPCR 試劑盒PanGreen PanProbes™ 一步驟 RT-qPCR 試劑盒。

逆轉錄酶 RScript Reverse Transcriptase / 50 Reactions| Bio-Helix 貨號RT001-0050

RScript Reverse Transcriptase / 50 Reactions

產品描述:

Overcome the most challenging RNA structures over a wide temperature range.

Bio-Helix RScript 逆轉錄酶(Bio-Helix RScript Reverse Transcriptase) – 專為研究和診斷應用而創新設計，滿足所有的 cDNA 合成需求，並克服寬溫度範圍內最具挑戰性的二級 RNA 結構( secondary RNA structures)。新一代工程重組 M-MLV 逆轉錄酶( M-MLV reverse transcriptase)具有更高的熱穩定性(thermostability)、持續合成能力(processivity)、穩健性(robustness)、最佳 cDNA 產量、用於降低 RNase H 活性的專有位點突變以及延長的半衰期，是世界上最通用的逆轉錄酶( versatile reverse transcriptase )不僅簡單地滿足常規 cDNA 合成要求，而且即使是手頭最具挑戰性的 RNA 樣品也能實現卓越的性能。

Figure 1. Stability of RScript reverse transcriptase:
The stability RT-qPCR data shows the performance of RScript reverse transcriptase is maintained without any significant alteration in performance at 37 °C after a period of 3 weeks (21 days), demonstrating its high resistance to temperature and time. The percentage of RScript reverse transcriptase activity was calculated by dividing values at each reaction temperature.

A: Bio-Helix RScript reverse transcriptase #RT001-0050/ RT001-0250
B: Promega Improm II #A3802
C: Thermo SuperScript III #18080093
D: SuperScript IV #18090010

Figure 2. Enhancement of the thermostability:
Reverse transcription of a 1 kb fragment ARHGAP29 RNA using RScript reverse transcriptase or competitor reverse transcriptases were used to carry this experiment. Regardless of the variations in temperatures (42 -60 °C) RScript reverse transcriptase shows a stable and superior performance at higher temperatures of up to 60 ℃ compared to other brands. The molecular weight marker used was DNA Ladder 1kb #SDL-1000R.

Figure 3. Elongation of RNA templates up to 11 kb in length :
RScript reverse transcriptase was used in a reaction with a range of human bladder cancer cell lines – 5637 (HTB-9) RNA. The resulting synthesized cDNA (8 kb ,11kb) was followed by PCR and visualized on a 2% agarose gel. The molecular weight marker used was DNA Ladder 1kb #SDL-1000R.

RScript Reverse Transcriptase 50 Reactions Bio-Helix 貨號RT001-0050

Figure 4. Performance of One-Step RT-qPCR with RScript reverse transcriptase :

To evaluate the RT performance for the COVID-19 fragments, the N1 gene with 100 copies per reaction was tested in the one-step RT-qPCR. Experiments proved to be feasible and effective (mean Ct : 34.88).

* COVID-19 fragments were originally obtained from Twist Synthetic SARS-CoV-2 RNA Controls (# EPI_ISL_1662307).

Red: Bio-HeliX RScript reverse transcriptase #RT001-0050
Green: Improm-II #A3802
Blue: Superscript IV #18090010
Figure 5. Performing RT-qPCR with RScript reverse transcriptase :
At a temperature of 55 °C, RScript reverse transcriptase exceeds performance demonstrating better efficiency and higher cDNA yields. RScript reverse transcriptase shows a stable and superior performance at 55 ℃ compared to other brands. Therefore RScript reverse transcriptase does not encounter significant higher Ct values compared to Improm II and SuperScript IV due to their low amounts of input cDNA.

*Promega Improm-II #A3802 is a registered trademark of Promega, Thermo Superscript-III #18080093 and Superscript-IV #18090010 are registered trademarks of Thermo Scientific. The trademark holders are not affiliated with Bio-Helix Co., Ltd. and do not endorse these products.

RScript cDNA 合成試劑盒(200 U/μl) | RScript cDNA Synthesis Kit貨號RK001-0050

RScript cDNA Synthesis Kit (50 Rxns)

Categories: RNA Reagent

一、產品介紹

RScript cDNA 合成試劑盒(RScript cDNA Synthesis Kit, RK001-0050) 專門用於研究和診斷應用，可滿足所有的 cDNA 合成需求；克服寬溫度範圍內最具挑戰性的二級 RNA 結構(secondary RNA structures)。套組內包含新一代工程重組 M-MLV 逆轉錄酶(M-MLV reverse transcriptase)，具有改進的熱穩定性( improved thermostability)、持續合成能力(robustness)、穩健性、最佳 cDNA 產量( optimal cDNA yields)、Moloney 小鼠白血病病毒逆轉錄酶 (M-MLV RT) 缺乏 DNA 核酸內切酶活性，具有較低的 RNase H 活性。是最通用的世界上的逆轉錄酶，不僅可以簡單地滿足常規 cDNA 合成要求，而且即使對於手頭最具挑戰性的 RNA 樣品也能實現卓越的性能。

Reverse transcription of a 330 bp fragment CCD1A RNA using RScript cDNA Synthesis Kit to carry this experiment. The PCR data shows that RScript cDNA Synthesis Kit have a stable and superior performance at higher temperatures.

二、產品說明

Template Total RNA, synthetic RNA transcript or poly(A)+mRNA, or the RNA should be avoided for cross-contamination with DNA.

Primer Selection Primer amounts recommended for efficient cDNA synthesis are 2.5 uM of oligo(dT) (anneal to the 3’-poly(A) + mRNA) or 2.5 ng/ul of random primers (anneal at non-specific sites of RNA templates), or 2 uM of genespecific primers per 20 ul reaction.

一步驟 RT-qPCR 試劑盒 | PanProbes™ One-Step RT-qPCR Kit Bio-Helix 貨號QPR01-0100

PanProbes™ One-Step RT-qPCR Kit

產品描述:

PanProbes™ 一步驟 RT-qPCR 試劑盒由於其 RScript 逆轉錄酶(RScript reverse transcriptase)、降低的 RNase H+ 活性 MMLV 酶以及強大的 RNase 抑制劑混合物(RNase inhibitors mix )而提供目標 RNA 水平的高靈敏度，旨在減少反應設置期間的 RNA 降解(RNA degradation)、錯誤引物(mispriming)、反轉錄(reverse transcription )以保證最佳的 RT 效率。Universal qPCR Master Mix 是一種 2x 濃縮的即用型預混液，針對基於探針的real-time PCR 進行了優化，並與大多數商用實時 real-time PCR systems (ROX-independent and ROX-dependent)兼容。它包含抗體介導的熱啟動 Taq DNA 聚合酶( antibody-mediated hot-start Taq DNA polymerase)、dNTP、MgCl2、增強劑(enhancers)、穩定劑(stabilizers)和成功進行 PCR 反應( success PCR reaction)的必需品。

* 增強的 RSCRIPT 逆轉錄酶與 RNASE 抑制劑。
* 針對基於探針的實時 PCR 進行了優化。
* 與大多數 QPCR 系統兼容。

Prepare (on ice or at room temperature) enough assay Master Mix for all reactions by adding all necessary components, except the RNA template, according to the recommendations in Table 1 .

3. Combine the assay Master Mix thoroughly to ensure consistency and equally dispense the solution into each qPCR tube or into the wells of a qPCR plate. Employ good pipetting practice to ensure assay precision and accuracy.

4. Add RNA template (and DNase-free H2O if needed) to the PCR tubes or wells containing assay Master Mix (Table 1), seal the tubes or wells with flat caps or optically transparent film. Note: to ensure thorough mixing of reaction components, vortex for approximately 30 seconds (or more).

5. Spin the tubes or plate to remove any air bubbles and collect the reaction mixture in the vessel bottom.

6. Setup the thermal cycling protocol on a real-time PCR instrument according to Table 2. Note: optimization may be needed for better performance.

7. Load the PCR tubes or plate into the real-time PCR instrument and commence the run. 8. Perform data analysis according to the instrument-specific instructions.

Process in the thermal cycler for 35~45 cycles as follows:

Figure 1. Performance of PanProbes™ One-Step RT-qPCR Master Mix

To evaluate the performance, the Ribonuclease P (RP)gene with varying RNA template concentrations, through serial dilutions, were used. Based on the fluorescence value of the RP probe, the results show the performance corresponding to the Ct value with different copy numbers.

即用型 qPCR 混合液PanGreen™ Universal SYBR® Green Master Mix| Bio-Helix 貨號QSD01-0100

PanGreen™ Universal SYBR® Green Master Mix

產品描述:

PanGreenTM Universal SYBR® Green Master Mix 是一種 2 倍濃縮的即用型 Master Mix 混合液，增強了基於染料的定量 PCR (dye-based quantitative PCR, qPCR) 並與大多數市售實時 PCR 系統（不依賴 ROX 和依賴 ROX）兼容。它包含 NanoTaq 熱啟動 DNA 聚合酶( NanoTaq hot-start DNA polymerase)、dNTP、MgCl2、SYBR® Green I 染料、增強劑、穩定劑和成功進行 PCR 反應的必需品。

* 增強型納米複合物介導的熱啟動酶 DNA 聚合酶(NANO COMPLEX-MEDIATED HOT START ENZYME DNA POLYMERASE)。
* 使用納米技術最大限度地提高產品產量。
* 非特異性條帶在反應過程中被消除。
* 與大多數 QPCR 系統兼容。

Figure 1. Performance of PanGreen™ Universal SYBR® Green Master Mix

A 500 bp human genomic DNA target was used to compare the two leading competing QPCR Master Mixes. All amplifications were performed in accordance with the manufacturer’s instructions. PanGreen™ Universal SYBR® Green Master Mix exhibited to be effective (mean Ct : 21.29) and highly specific because no second amplification signal could be identified with the melting curve .The results show that it has the best performance as compared with other QPCR Master Mix suppliers.

PanGreen™ Universal SYBR® Green Master Mix表現出有效（平均Ct：21.29）和高度特異性

RK001-0050 cDNA合成KIT	RScript™ cDNA Synthesis Kit (新一代工程重組 M-MLV 逆轉錄酶(M-MLV reverse transcriptase)) RScript cDNA 合成試劑盒(200 U/μl)	50 rxns
RT001-0050 RT逆轉錄酶	RScript™ Reverse Transcriptase 10,000U (新一代工程重組 M-MLV 逆轉錄酶(M-MLV reverse transcriptase)) 逆轉錄酶 RScript Reverse Transcriptase	50 rxns 10,000U/50μl
RT001-0250 RT逆轉錄酶	RScript™ Reverse Transcriptase 50,000U (新一代工程重組 M-MLV 逆轉錄酶(M-MLV reverse transcriptase)) 逆轉錄酶RScript Reverse Transcriptase	250 rxns 50,000U/250μl
RI001-0125 RNase Inhibitor	RIBOAssure™ RNase Inhibitor 5,000U (抑制真核酶如 RNase A、RNase B 和 RNase C 所表現出的核糖核酸酶 (RNase) 活性) RNase 抑制劑	125 rxns 5,000U/125μl
QPR01-0100 (Probe)	PanProbes™ One-Step RT-qPCR Kit (Probe) (新一代工程重組 M-MLV 逆轉錄酶(M-MLV reverse transcriptase)) 強大的 RNase 抑制劑混合物(RNase inhibitors mix) 增強的抗體介導的熱啟動 DNA 聚合酶 (針對基於探針的real-time PCR 進行了優化) 一步驟 RT-qPCR 試劑盒	100 reactions (20 μl vol)
QPD01-0100 (Probe)	PanProbes™ Universal qPCR MasterMix (針對基於probe- PCR (probe-based real-time PCR )進行了優化) 增強的抗體介導的熱啟動 DNA 聚合酶 PanProbes™ Universal qPCR預混液	100 reactions (20 μl vol)
QSR01-0100 (SYBR® Green)	PanGreen™ One-Step RT-qPCR Kit (SYBR® Green) (新一代工程重組 M-MLV 逆轉錄酶(M-MLV reverse transcriptase)) 強大的 RNase 抑制劑混合物(RNase inhibitors mix) 增強的抗體介導的熱啟動 DNA 聚合酶 Universal SYBR® Green Master Mix 2 倍濃縮的即用型混合液 Master Mix 一個步驟RT-qPCR 試劑盒PanGreen	100 reactions (20 μl vol)
QSD01-0100 SYBR® Green)	PanGreen™ Universal SYBR® Green Mastermix (SYBR® Green) (2 倍濃縮的即用型 Master Mix 混合液) 增強的抗體介導的熱啟動 DNA 聚合酶即用型 qPCR 混合液PanGreen	100 reactions (20 μl vol)
PDR03-0100	Total RNA Isolation Reagent (Reagent Based) for the tissue, cultured animal and bacterial cells, blood, and serum. Total RNA 分離試劑 (Reagent Based）	100 rxns