Maxima Reverse Transcriptases

Thermo Scientific Maxima反转录酶通过分子改造而得到,可实现最高的cDNA合成性能。我们的专利技术引入多种有利的突变,显著改善了这些酶的热稳定性、扩增效率和持续合成能力,从而提高cDNA合成性能。Maxima反转录酶有多种形式,可用于RT-PCR和RT-qPCR,包括单酶、经过优化的试剂盒和极其方便的预混液。Maxima反转录酶试剂盒和预混液还具有完整的gDNA去除步骤,工作流程简单高效。

点此订购

 下载Maxima H Minus cDNA合成预混液宣传页
 下载Maxima反转录酶宣传页

Developed through molecular evolution, our latest Maxima H Minus reverse transcriptase is an enzyme with diminished RNase H activity but contains all the features of Maxima RT with high thermostability and enhanced processivity.  With these improved features, Maxima H Minus reverse transcriptase offers a robust performance in first strand cDNA synthesis.

Order now    Request a sample

Feature video: Maxima first strand cDNA synthesis kits in action

Thermo Scientific Maxima First Strand cDNA Synthesis Kits with double-stranded DNase combine genomic DNA elimination and cDNA synthesis into a simple, one tube workflow that can be completed in as few as 15 minutes.

Maxima H Minus RTs are engineered with molecular evolution techniques selecting for mutations that confer dramatically improved thermostability, processivity and robust activity rates compared with wild-type M-MuLV enzymes. These features support higher overall cDNA yields with a variety of templates and improved synthesis from templates with complex secondary structures.

Maxima H Minus RT also shows greater efficiency with synthesis of full length cDNA templates. It lacks RNase H activity that normally degrades RNA from RNA-DNA duplexes as the cDNA is being synthesized; which helps to prevent premature degradation of RNA. In RT-qPCR applications, Maxima H Minus RT also enables high efficiency synthesis over a wide range of input template amounts providing sensitive and accurate quantification of cDNA.

High thermostability

Maxima H Minus Reverse Transcriptase outperforms other reverse transcriptases, producing high yields of full-length cDNA over a wide temperature range (Figure 1). Its tolerance of high reaction temperatures allows for efficient transcription of RNA regions with extensive secondary structure and helps to improve primer specificity increasing overall yields.

High yields of cDNA over a broad temperature range
Figure 1. High yields of cDNA over a broad temperature range. cDNA synthesis was performed using 1 μg of Invitrogen Millennium RNA markers (poly(A)-tailed) with oligo(dT)18 primers and (A) Maxima H Minus RT (20U) following the recommended  protocol across a temperature range (42 °C; 50 °C; 55 °C; 60 °C; 65 °C).  Comparable reactions were performed with RTs from other suppliers including (B) Takara PrimeScript RT, (C) Promega GoScript RT, and (D) NEB ProtoScript II RT, according to each manufacturer’s protocol. Reaction products were resolved by alkaline gel electrophoresis.

High processivity

Designed with proprietary mutations for enhanced processivity, Maxima H Minus RT has 50x increase in processivity compared with wild type MMuLV RT enzymes. This RT is capable of synthesizing full-length cDNA from a wide range of RNA templates (Figure 2).

Amplification of targets up to 20 kb in two-step RT-PCR
Figure 2. Amplification of targets up to 20 kb in two-step RT-PCR. Total RNA (1 μg) from human cells (lanes 1 and 2) or mouse cells (lanes 3 and 4) were used in a RT reaction with Maxima H Minus RT following the maufacturer’s recommended protocol. The resulting cDNA products were used as a template for PCR. M: Thermo Scientific GeneRuler 1 kb Plus DNA Ladder.

Efficient cDNA synthesis

Maxima H Minus RT is capable of efficient cDNA synthesis from a wide range of template amounts, outperforming other suppliers’ RTs with higher yields and better linearity in cDNA synthesis making it an ideal choice for RT-qPCR experiments (Figure 3). The premixed solutions in the Thermo Scientific Maxima First Strand cDNA Synthesis Kits help further improve reproducibility and save time during reaction setup.

Consistently efficient RT over a wide range of input RNA amounts
Figure 3. Consistently efficient RT over a wide range of input RNA amounts. Maxima H Minus First Strand cDNA Synthesis kit demonstrates consistently better reverse transcription efficiency than other suppliers’ kits. Amplification plots show variation of log (ΔRn) with PCR cycle number. RT-qPCR of human β-2 macroglobulin gene was performed from 10-fold serial dilutions of HeLa total RNA (1 μg to 1 pg). First strand cDNA was generated using the Maxima H Minus First Strand cDNA Synthesis kit and 7 other commercial First Strand cDNA Synthesis kits. cDNA was amplified using TaqMan Universal Master Mix II, with UNG on the Applied Biosystems ViiA7 Real-Time PCR System.

Maxima H Minus RT has been formulated into a convenient one-tube master mix with a simplified protocol that enables consistency and maximum control of your RT-qPCR.

Linearity over a broad dynamic range

The Maxima H Minus cDNA Synthesis Master Mix maintains high transcription efficiency and good linearity across a broad range of template concentrations (Figure 4). The observed linearity strongly suggests reliable representation of relative quantities of different transcripts when using large or small amounts of input RNA.

Figure 4. Broad dynamic range of Maxima H Minus cDNA Synthesis Master Mix. The standard curve illustrates high linearity (R2 = 0.999) across a broad range of input RNA, suggesting that the relative representation of specific RNA transcripts is preserved in the cDNA pool regardless of the abundance of total RNA. Amplification of the human 18S RNA gene was performed on 10-fold serial dilutions of HeLa total RNA (1 μg to 0.1 pg). First-strand cDNA was generated using the Maxima H Minus cDNA Synthesis Master Mix. cDNA was amplified using the Thermo Scientific Luminaris Probe qPCR Master Mix, low ROX, on the Applied Biosystems ViiA 7 Real-Time PCR System.

 

Consistent transcription efficiency

The Maxima H Minus cDNA Synthesis Master Mix offers higher efficiency than RTs from other suppliers at low and high input RNA amounts (Figure 5). The higher transcription efficiency allows the use of less RNA and accurate detection of less expressed transcripts.

Enhanced transcription efficiency of Maxima H Minus cDNA Synthesis Master Mix

Figure 5. Enhanced transcription efficiency of Maxima H Minus cDNA Synthesis Master Mix. The Maxima H Minus cDNA Synthesis Master Mix demonstrates better efficiency than other suppliers’ RTs over a wide range of input RNA amounts. Amplification of the human 18S RNA gene was performed on 10-fold serial dilutions of HeLa total RNA (1 μg to 0.1 pg). First-strand cDNA was generated using the Maxima H Minus cDNA Synthesis Master Mix, Thermo Scientific Maxima First Strand cDNA Synthesis Kit for RT-qPCR, and RTs from four other suppliers. cDNA was amplified using the Luminaris Probe qPCR Master Mix, low ROX on the ViiA 7 Real-Time PCR System. Amplification plots indicate variation of ΔRn with cycle number.

Reliable transcription across an array of targets in a 96-gene panel

The Maxima H Minus cDNA Synthesis Master Mix shows consistently better efficiency than RT master mixes from other suppliers over a range of 96 target genes in RT-qPCR when normalized to data generated with the Maxima First Strand cDNA Synthesis Kit for RT-qPCR (Figure 6).

Figure 6. Consistently lower Ct values. Maxima H Minus cDNA Synthesis Master Mix shows higher cDNA synthesis efficiency compared to other commercial master mixes over a wide range of targets. Maxima H Minus cDNA Synthesis Master Mix was compared to Thermo Scientific Maxima First Strand cDNA Synthesis Kit for RT-qPCR and master mixes from other suppliers using 96-gene Applied Biosystems TaqMan Assay panels with 100 ng HeLa total RNA input. Using the Maxima First Strand cDNA Synthesis Kit for RT-qPCR as the reference, the ΔCt values (ΔCt = Ct Maxima H Minus cDNA Synthesis Master Mix or other commercial product – CtMaxima First Strand cDNA Synthesis Kit for RT-qPCR) are shown for each of the 96 genes in the panel.
 

Easy, integrated gDNA removal step

The Maxima H Minus cDNA Synthesis Master Mix is available with a double-strand–specific DNase (dsDNase), which enables faster, more efficient, and complete gDNA removal compared with conventional DNase I. dsDNase-treated RNA samples show no decrease in RNA integrity or quantity.  With the use of dsDNase treatment prior to cDNA synthesis, you can minimize the risk of sample loss observed following sample clean-up with conventional DNase I treatment.

Maxima H Minus RTs are engineered with molecular evolution techniques selecting for mutations that confer dramatically improved thermostability, processivity and robust activity rates compared with wild-type M-MuLV enzymes. These features support higher overall cDNA yields with a variety of templates and improved synthesis from templates with complex secondary structures.

Maxima H Minus RT also shows greater efficiency with synthesis of full length cDNA templates. It lacks RNase H activity that normally degrades RNA from RNA-DNA duplexes as the cDNA is being synthesized; which helps to prevent premature degradation of RNA. In RT-qPCR applications, Maxima H Minus RT also enables high efficiency synthesis over a wide range of input template amounts providing sensitive and accurate quantification of cDNA.

High thermostability

Maxima H Minus Reverse Transcriptase outperforms other reverse transcriptases, producing high yields of full-length cDNA over a wide temperature range (Figure 1). Its tolerance of high reaction temperatures allows for efficient transcription of RNA regions with extensive secondary structure and helps to improve primer specificity increasing overall yields.

High yields of cDNA over a broad temperature range
Figure 1. High yields of cDNA over a broad temperature range. cDNA synthesis was performed using 1 μg of Invitrogen Millennium RNA markers (poly(A)-tailed) with oligo(dT)18 primers and (A) Maxima H Minus RT (20U) following the recommended  protocol across a temperature range (42 °C; 50 °C; 55 °C; 60 °C; 65 °C).  Comparable reactions were performed with RTs from other suppliers including (B) Takara PrimeScript RT, (C) Promega GoScript RT, and (D) NEB ProtoScript II RT, according to each manufacturer’s protocol. Reaction products were resolved by alkaline gel electrophoresis.

High processivity

Designed with proprietary mutations for enhanced processivity, Maxima H Minus RT has 50x increase in processivity compared with wild type MMuLV RT enzymes. This RT is capable of synthesizing full-length cDNA from a wide range of RNA templates (Figure 2).

Amplification of targets up to 20 kb in two-step RT-PCR
Figure 2. Amplification of targets up to 20 kb in two-step RT-PCR. Total RNA (1 μg) from human cells (lanes 1 and 2) or mouse cells (lanes 3 and 4) were used in a RT reaction with Maxima H Minus RT following the maufacturer’s recommended protocol. The resulting cDNA products were used as a template for PCR. M: Thermo Scientific GeneRuler 1 kb Plus DNA Ladder.

Efficient cDNA synthesis

Maxima H Minus RT is capable of efficient cDNA synthesis from a wide range of template amounts, outperforming other suppliers’ RTs with higher yields and better linearity in cDNA synthesis making it an ideal choice for RT-qPCR experiments (Figure 3). The premixed solutions in the Thermo Scientific Maxima First Strand cDNA Synthesis Kits help further improve reproducibility and save time during reaction setup.

Consistently efficient RT over a wide range of input RNA amounts
Figure 3. Consistently efficient RT over a wide range of input RNA amounts. Maxima H Minus First Strand cDNA Synthesis kit demonstrates consistently better reverse transcription efficiency than other suppliers’ kits. Amplification plots show variation of log (ΔRn) with PCR cycle number. RT-qPCR of human β-2 macroglobulin gene was performed from 10-fold serial dilutions of HeLa total RNA (1 μg to 1 pg). First strand cDNA was generated using the Maxima H Minus First Strand cDNA Synthesis kit and 7 other commercial First Strand cDNA Synthesis kits. cDNA was amplified using TaqMan Universal Master Mix II, with UNG on the Applied Biosystems ViiA7 Real-Time PCR System.

Maxima H Minus RT has been formulated into a convenient one-tube master mix with a simplified protocol that enables consistency and maximum control of your RT-qPCR.

Linearity over a broad dynamic range

The Maxima H Minus cDNA Synthesis Master Mix maintains high transcription efficiency and good linearity across a broad range of template concentrations (Figure 4). The observed linearity strongly suggests reliable representation of relative quantities of different transcripts when using large or small amounts of input RNA.

Figure 4. Broad dynamic range of Maxima H Minus cDNA Synthesis Master Mix. The standard curve illustrates high linearity (R2 = 0.999) across a broad range of input RNA, suggesting that the relative representation of specific RNA transcripts is preserved in the cDNA pool regardless of the abundance of total RNA. Amplification of the human 18S RNA gene was performed on 10-fold serial dilutions of HeLa total RNA (1 μg to 0.1 pg). First-strand cDNA was generated using the Maxima H Minus cDNA Synthesis Master Mix. cDNA was amplified using the Thermo Scientific Luminaris Probe qPCR Master Mix, low ROX, on the Applied Biosystems ViiA 7 Real-Time PCR System.

 

Consistent transcription efficiency

The Maxima H Minus cDNA Synthesis Master Mix offers higher efficiency than RTs from other suppliers at low and high input RNA amounts (Figure 5). The higher transcription efficiency allows the use of less RNA and accurate detection of less expressed transcripts.

Enhanced transcription efficiency of Maxima H Minus cDNA Synthesis Master Mix

Figure 5. Enhanced transcription efficiency of Maxima H Minus cDNA Synthesis Master Mix. The Maxima H Minus cDNA Synthesis Master Mix demonstrates better efficiency than other suppliers’ RTs over a wide range of input RNA amounts. Amplification of the human 18S RNA gene was performed on 10-fold serial dilutions of HeLa total RNA (1 μg to 0.1 pg). First-strand cDNA was generated using the Maxima H Minus cDNA Synthesis Master Mix, Thermo Scientific Maxima First Strand cDNA Synthesis Kit for RT-qPCR, and RTs from four other suppliers. cDNA was amplified using the Luminaris Probe qPCR Master Mix, low ROX on the ViiA 7 Real-Time PCR System. Amplification plots indicate variation of ΔRn with cycle number.

Reliable transcription across an array of targets in a 96-gene panel

The Maxima H Minus cDNA Synthesis Master Mix shows consistently better efficiency than RT master mixes from other suppliers over a range of 96 target genes in RT-qPCR when normalized to data generated with the Maxima First Strand cDNA Synthesis Kit for RT-qPCR (Figure 6).

Figure 6. Consistently lower Ct values. Maxima H Minus cDNA Synthesis Master Mix shows higher cDNA synthesis efficiency compared to other commercial master mixes over a wide range of targets. Maxima H Minus cDNA Synthesis Master Mix was compared to Thermo Scientific Maxima First Strand cDNA Synthesis Kit for RT-qPCR and master mixes from other suppliers using 96-gene Applied Biosystems TaqMan Assay panels with 100 ng HeLa total RNA input. Using the Maxima First Strand cDNA Synthesis Kit for RT-qPCR as the reference, the ΔCt values (ΔCt = Ct Maxima H Minus cDNA Synthesis Master Mix or other commercial product – CtMaxima First Strand cDNA Synthesis Kit for RT-qPCR) are shown for each of the 96 genes in the panel.
 

Easy, integrated gDNA removal step

The Maxima H Minus cDNA Synthesis Master Mix is available with a double-strand–specific DNase (dsDNase), which enables faster, more efficient, and complete gDNA removal compared with conventional DNase I. dsDNase-treated RNA samples show no decrease in RNA integrity or quantity.  With the use of dsDNase treatment prior to cDNA synthesis, you can minimize the risk of sample loss observed following sample clean-up with conventional DNase I treatment.

Ordering information

申请预混液免费样品  申请单酶或试剂盒免费样品

在2步法RT-qPCR中高效完成反转录

我们全新的Thermo Scientific Maxima H Minus cDNA合成预混液可在2步法RT-qPCR中提供始终如一的高效和可重复的反转录。为了给您的cDNA合成带来最高价值,这种方便的单管式预混液配方中加入了一种工程化RNase H酶——Maxima H Minus反转录酶——其简单的方案可为您的RT-qPCR带来一致性,并实现最大控制。

为什么使用预混液?

  • 反应组装更方便
  • 有助于减少多个步骤带来的移液差异,提高RT-qPCR数据的准确性
  • 降低样品之间发生交叉污染的风险

为什么使用Maxima H Minus反转录酶?

  • 是通过分子改造得到的工程化MMuLV(莫洛尼鼠白血病病毒)反转录酶
  • 更高热稳定性
  • 更快合成速率
  • 持续合成能力提高50倍
  • 无RNase H活性
  • 已证明比竞争产品的灵敏度更高

在较宽的动态范围内具有高转录效率

Maxima H Minus cDNA合成预混液可在较宽的模板浓度范围内保持高转录效率和良好的线性(图1)。良好的线性表明,无论使用大量还是少量起始RNA,都能可靠代表不同转录物的相对数量。

Broad dynamic range of Maxima H Minus cDNA Synthesis Master Mix


图1. Maxima H Minus cDNA合成预混液的广泛动态范围。
标准曲线在宽的起始RNA范围内具有高度线性度(R2 = 0.999),这表明无论总RNA丰度是多少,特定RNA转录物的相对代表性在cDNA库中得以保留。将HeLa细胞总RNA进行10倍连续稀释(1 μg至0.1 pg),然后对人18S RNA基因进行扩增。使用Maxima H Minus cDNA合成预混液生成第一链cDNA。使用Thermo Scientific Luminaris Probe qPCR预混液(低ROX),在Applied Biosystems ViiA 7实时PCR系统上扩增cDNA。

 

一致的转录效率

在使用少量和大量起始RNA时,Maxima H Minus cDNA合成预混液都比其他品牌的反转录酶具有更高的效率(图2)。转录效率越高,RNA使用量越少,并且能够准确检测低表达的转录物。

图2. Maxima H Minus cDNA合成预混液的转录效率更强。在广泛的RNA起始量范围内,Maxima H Minus cDNA合成预混液比其他品牌的反转录酶具有更高的效率。将HeLa细胞总RNA进行10倍连续稀释(1 μg to 0.1 pg),然后对人18S RNA基因进行扩增。使用Maxima H Minus cDNA合成预混液、用于RT-qPCR 的Thermo Scientific Maxima第一链cDNA合成试剂盒以及来自其他四家供应商的反转录酶,生成第一链cDNA。使用Luminaris Probe qPCR预混液(低ROX),在ViiA 7实时PCR系统上扩增cDNA。扩增曲线表示ΔRn随循环数的变化。

对靶标阵列实现一致、可靠的转录

使用Maxima第一链cDNA合成试剂盒得到的RT-qPCR数据进行归一化处理后发现,在RT-qPCR中96个靶基因中,Maxima H Minus cDNA合成预混液始终比其他供应商的反转录酶预混液具有更高的效率(图3)。

图3. Ct值一致降低。在广泛的靶标范围内,Maxima H Minus cDNA合成预混液比其他市售预混液具有更高的cDNA合成效率。使用96基因Applied Biosystems TaqMan®检测板和100 ng HeLa总RNA起始量,将Maxima H Minus cDNA合成预混液与Thermo Scientific Maxima First Strand cDNA Synthesis Kit for RT-qPCR以及其他品牌的预混液进行比较。以Maxima First Strand cDNA Synthesis Kit for RT-qPCR作为对照,得到检测板中96个基因的ΔCt值(ΔCt = Maxima H Minus cDNA 合成预混液或其他市售产品的Ct – 用于RT-qPCR 的Maxima First Strand cDNA Synthesis Kit for RT-qPCR的Ct)。

产品特点:

  • 在宽的RNA起始量范围内,RT-qPCR结果的一致性更高,cDNA合成效率更高
  • 方便的预混液形式,可降低移液差异
  • 包含用于RT-qPCR的dsDNase和no-RT对照,可控制RNA制备中的gDNA污染风险

 

技术细节:

  • 在96-基因检测板阵列中,cDNA合成效率始终较高
  • 在较宽的动态范围(1 pg-1 μg的总RNA)内,cDNA合成呈更高线性度
  • 在42°C-55°C的反转录过程中保持完整的酶活性

 

完整的cDNA合成工作流程解决方案

Maxima H Minus cDNA合成预混液具有双链特异性DNA酶(dsDNase),与传统DNase I相比,可更快、更高效和更完全地去除gDNA。dsDNase处理的RNA样品未显示出RNA完整性和数量的降低。在cDNA合成之前使用dsDNase处理,可最大限度降低使用传统DNase I净化样品带来的损失风险。

Maxima No-RT 对照混合物将与Maxima H Minus反转录酶预混液一起提供,从而为2步法RT-qPCR提供完整的cDNA合成解决方案。这种No-RT 对照混合物包含反转录预混液中除Maxima H Minus反转录酶以外的所有成分,使研究人员能够准确确定无残留的gDNA污染。

Maxima和Maxima H Minus反转录酶的分子改造

Thermo Scientific Maxima和Maxima H Minus反转录酶是利用体外分子改造技术而获得,该技术通过向传统M-MuLV反转录酶中引入多种突变,并从中筛选出能够改善cDNA合成性能的突变体.。改善的性能包括:

  • 更高的全长cDNA得率
  • 高反应温度,提高转录性能
  • 对长RNA模板的高转录效率
     

在广泛温度范围内的全长cDNA

在广泛的温度范围内,Maxima反转录酶优于其他反转录酶。它们对高反应温度的耐受性使其能够对具有广泛二级结构的RNA区域进行高效转录,并且有助于提高引物特异性,提高全长DNA的得率(图4)。

High yields of cDNA over a broad temperature range

 

图4. 在广泛温度范围内的高cDNA得率。利用1 μg的Invitrogen Millennium RNA标记物(带有poly(A)-结构)和oligo(dT)18引物,使用Maxima H Minus反转录酶和来自其他供应商的反转录酶,按照说明书完成含有放射性标记cDNA合成,并进行对比。在碱性琼脂糖凝胶上分离反应产物。

长片断RT-PCR的扩增能力增强

Maxima H Minus反转录酶采用专利突变设计改善性能,能够从非常长的RNA模板合成全长cDNA(图5)。

Amplification of long targets in two-step RT-PCR

 

图5. 对两步法RT-PCR中长靶标的扩增。使用Maxima H Minus反转录酶和来自其他供应商的反转录酶,根据产品说明书以哺乳动物细胞的总RNA(1ug)进行重复反转录反应。将所得cDNA产物作为模板进行PCR扩增,并在琼脂糖凝胶上进行分离和观察。仅Maxima H Minus反转录酶能够生成非常长(13.3kb)的产物,并且具有高得率。

RT-qPCR的理想选择-- 灵敏度高和可重复性定量

Maxima反转录酶能够在较宽的模板量范围内完成可重复的cDNA合成,使其成为RT-qPCR实验的理想选择(图6)。Thermo Scientific Maxima第一链cDNA合成试剂盒中的预混液有助于进一步改善重复性,并节省反应组装过程的时间。

 

Reproducible cDNA synthesis and low variability with a wide range of starting RNA amounts

图6. 在较宽的起始RNA量范围内,实现可重复的cDNA合成和低变异率(<1% SD/Ct)。在16个重复反应中,使用Maxima第一链cDNA合成试剂盒,以100、10、1、0.1、0.01和0.001 ng哺乳动物细胞总RNA合成第一链cDNA。以合成的cDNA为模板,使用Themo Scientific Maxima SYBR Green/ROX qPCR预混液进行qPCR扩增。扩增曲线图说明,在起始用量范围内具有良好的一致性。

具有完整的gDNA去除步骤,工作流程简单

Maxima第一链cDNA试剂盒包括双链特异性DNA酶(dsDNase),能够在cDNA合成前清除基因组DNA,无需任何中间样品纯化步骤。经过改造的dsDNase能够在2分钟内去除污染的基因组DNA,并且不会损害RNA数量或质量,也不会降解引物和探针等单链DNA(图7)。温和的热处理(55℃)即可轻松使dsDNase失活。所得无基因组DNA的RNA样品可直接用于cDNA合成反应,大大简化了实验工作流程。

Effective&#x20;removal&#x20;of&#x20;genomic&#x20;DNA&#x20;using&#x20;dsDNase

图7. 使用dsDNase有效去除基因组DNA。利用多种cDNA合成试剂盒,在有(RT +)和无(RT-)反转录酶的情况下对PBGD基因进行两步法RT-qPCR分析并绘图。使用具有dsDNase的Maxima第一链cDNA合成试剂盒或具有gDNA去除功能的其他供应商试剂盒,以0.2 ng总Jurkat RNA进行cDNA合成。使用Maxima试剂盒得到橙色、平坦的反转录反应曲线表明完全去除了污染的gDNA,而使用其他供应商试剂盒得到的反转录反应曲线显示具有残留gDNA的扩增。

在广泛的温度范围内能够有效保护RNA

RNA酶抑制剂通常会在高温条件下变性,释放RNA酶回到反应混合物中,随后损害和降解RNA。为了避免RNA被RNA酶破坏,所有Maxima第一链cDNA合成试剂盒均包含经改造的热稳定型Thermo Scientific RiboLock RNA酶抑制剂。蛋白质被“锁定”在RNA酶上,在高达55℃的温度下也能抑制RNA降解(图8)。RiboLock RNA酶抑制剂的高度热稳定性,对于在高反应温度下成功完成反转录至关重要。

RiboLock&#x20;RNase&#x20;Inhibitor&#x20;efficiently&#x20;protects&#x20;RNA&#x20;and&#x20;inhibits&#x20;up&#x20;to&#x20;2&#x20;ng&#x2f;20&#x20;&mu;L&#x20;of&#x20;RNase&#x20;A

图8. RiboLock RNA酶抑制剂可有效保护RNA并抑制高达2 ng/20 μL的RNA酶A。在37°C下,向人总RNA(1μg)分装样品(20 μL)中加入20 U的RiboLock RNA酶抑制剂,并提高RNase A的含量。
M:Thermo Scientific RiboRuler高分子量范围RNA Ladder,即用型
C:人总RNA
1:含RNAase A的人总RNA
2-5:含RiboLock RNA酶抑制剂和RNase A的人总RNA

RiboLock RNA酶抑制剂具有高度热稳定性。向人总RNA(1μg)分装样品(20 μL)中加入20 U的RiboLock RNA酶抑制剂和50 pg的RNase A,并在升高的温度下孵育。

 

了解分子进化

对聚合酶精细结构和功能的了解有限, 使我们使用合理的设计标准提高酶性能的想法受到了限制。通过模拟天然特性并使用定向进化来改善酶的性质,能够打破这种限制。

我们的专利技术——区室化核糖体展示(CRO)——能够实现快速、高效的反转录酶体外改进。该技术已经在野生型MMuLV反转录酶中引入和选择出多个有利突变,生成新型高度热稳定和高合成能力反转录酶,取代了相应的野生型反转录酶。

通过CRD技术进行分子改造包括几个步骤(图9)。首先,从野生型MMuLV反转录酶基因(1)开始,基于随机诱变创建mRNA文库(2)。接下来,在体外将mRNA文库翻译成与其mRNA前体相关的蛋白质(3)。然后,将蛋白质-mRNA复合物置于反转录酶反应混合物中并乳化,得到含有一个蛋白质-mRNA复合物的不同区室。最后,升高温度以产生选择性压力,在该选择压力下,只有改良突变体能够存活并产生全长cDNA(4)。通过结合性能最佳的突变,构建了高度进行性MMuLV RT突变体,其能够在高温下合成全长cDNA。

  1. Baranauskas A et al. (2012) Generation and characterization of new highly thermostable and processive M-MuLV reverse transcriptase variants. PEDS. doi:10.1093/protein/gzs034
  2. Skirgaila R et al. (2013) Compartmentalization of destabilized enzyme–mRNA–ribosome complexes generated by ribosome display: a novel tool for the directed evolution of enzymes. PEDS. doi:10.1093/protein/gzt017
     
Key&#x20;steps&#x20;in&#x20;the&#x20;molecular&#x20;evolution&#x20;of&#x20;MMuLV&#x20;RT

图9. MmuLV反转录酶分子改造的关键步骤

Comparison&#x20;of&#x20;RTs&#x20;and&#x20;cDNA&#x20;synthesis&#x20;with&#x20;or&#x20;without&#x20;functional&#x20;RNase&#x20;H&#x20;activity

Figure 10. Comparison of RTs and cDNA synthesis with or without functional RNase H activity.

在2步法RT-qPCR中高效完成反转录

我们全新的Thermo Scientific Maxima H Minus cDNA合成预混液可在2步法RT-qPCR中提供始终如一的高效和可重复的反转录。为了给您的cDNA合成带来最高价值,这种方便的单管式预混液配方中加入了一种工程化RNase H酶——Maxima H Minus反转录酶——其简单的方案可为您的RT-qPCR带来一致性,并实现最大控制。

为什么使用预混液?

  • 反应组装更方便
  • 有助于减少多个步骤带来的移液差异,提高RT-qPCR数据的准确性
  • 降低样品之间发生交叉污染的风险

为什么使用Maxima H Minus反转录酶?

  • 是通过分子改造得到的工程化MMuLV(莫洛尼鼠白血病病毒)反转录酶
  • 更高热稳定性
  • 更快合成速率
  • 持续合成能力提高50倍
  • 无RNase H活性
  • 已证明比竞争产品的灵敏度更高

在较宽的动态范围内具有高转录效率

Maxima H Minus cDNA合成预混液可在较宽的模板浓度范围内保持高转录效率和良好的线性(图1)。良好的线性表明,无论使用大量还是少量起始RNA,都能可靠代表不同转录物的相对数量。

Broad&#x20;dynamic&#x20;range&#x20;of&#x20;Maxima&#x20;H&#x20;Minus&#x20;cDNA&#x20;Synthesis&#x20;Master&#x20;Mix


图1. Maxima H Minus cDNA合成预混液的广泛动态范围。
标准曲线在宽的起始RNA范围内具有高度线性度(R2 = 0.999),这表明无论总RNA丰度是多少,特定RNA转录物的相对代表性在cDNA库中得以保留。将HeLa细胞总RNA进行10倍连续稀释(1 μg至0.1 pg),然后对人18S RNA基因进行扩增。使用Maxima H Minus cDNA合成预混液生成第一链cDNA。使用Thermo Scientific Luminaris Probe qPCR预混液(低ROX),在Applied Biosystems ViiA 7实时PCR系统上扩增cDNA。

 

一致的转录效率

在使用少量和大量起始RNA时,Maxima H Minus cDNA合成预混液都比其他品牌的反转录酶具有更高的效率(图2)。转录效率越高,RNA使用量越少,并且能够准确检测低表达的转录物。

图2. Maxima H Minus cDNA合成预混液的转录效率更强。在广泛的RNA起始量范围内,Maxima H Minus cDNA合成预混液比其他品牌的反转录酶具有更高的效率。将HeLa细胞总RNA进行10倍连续稀释(1 μg to 0.1 pg),然后对人18S RNA基因进行扩增。使用Maxima H Minus cDNA合成预混液、用于RT-qPCR 的Thermo Scientific Maxima第一链cDNA合成试剂盒以及来自其他四家供应商的反转录酶,生成第一链cDNA。使用Luminaris Probe qPCR预混液(低ROX),在ViiA 7实时PCR系统上扩增cDNA。扩增曲线表示ΔRn随循环数的变化。

对靶标阵列实现一致、可靠的转录

使用Maxima第一链cDNA合成试剂盒得到的RT-qPCR数据进行归一化处理后发现,在RT-qPCR中96个靶基因中,Maxima H Minus cDNA合成预混液始终比其他供应商的反转录酶预混液具有更高的效率(图3)。

图3. Ct值一致降低。在广泛的靶标范围内,Maxima H Minus cDNA合成预混液比其他市售预混液具有更高的cDNA合成效率。使用96基因Applied Biosystems TaqMan®检测板和100 ng HeLa总RNA起始量,将Maxima H Minus cDNA合成预混液与Thermo Scientific Maxima First Strand cDNA Synthesis Kit for RT-qPCR以及其他品牌的预混液进行比较。以Maxima First Strand cDNA Synthesis Kit for RT-qPCR作为对照,得到检测板中96个基因的ΔCt值(ΔCt = Maxima H Minus cDNA 合成预混液或其他市售产品的Ct – 用于RT-qPCR 的Maxima First Strand cDNA Synthesis Kit for RT-qPCR的Ct)。

产品特点:

  • 在宽的RNA起始量范围内,RT-qPCR结果的一致性更高,cDNA合成效率更高
  • 方便的预混液形式,可降低移液差异
  • 包含用于RT-qPCR的dsDNase和no-RT对照,可控制RNA制备中的gDNA污染风险

 

技术细节:

  • 在96-基因检测板阵列中,cDNA合成效率始终较高
  • 在较宽的动态范围(1 pg-1 μg的总RNA)内,cDNA合成呈更高线性度
  • 在42°C-55°C的反转录过程中保持完整的酶活性

 

完整的cDNA合成工作流程解决方案

Maxima H Minus cDNA合成预混液具有双链特异性DNA酶(dsDNase),与传统DNase I相比,可更快、更高效和更完全地去除gDNA。dsDNase处理的RNA样品未显示出RNA完整性和数量的降低。在cDNA合成之前使用dsDNase处理,可最大限度降低使用传统DNase I净化样品带来的损失风险。

Maxima No-RT 对照混合物将与Maxima H Minus反转录酶预混液一起提供,从而为2步法RT-qPCR提供完整的cDNA合成解决方案。这种No-RT 对照混合物包含反转录预混液中除Maxima H Minus反转录酶以外的所有成分,使研究人员能够准确确定无残留的gDNA污染。

Maxima和Maxima H Minus反转录酶的分子改造

Thermo Scientific Maxima和Maxima H Minus反转录酶是利用体外分子改造技术而获得,该技术通过向传统M-MuLV反转录酶中引入多种突变,并从中筛选出能够改善cDNA合成性能的突变体.。改善的性能包括:

  • 更高的全长cDNA得率
  • 高反应温度,提高转录性能
  • 对长RNA模板的高转录效率
     

在广泛温度范围内的全长cDNA

在广泛的温度范围内,Maxima反转录酶优于其他反转录酶。它们对高反应温度的耐受性使其能够对具有广泛二级结构的RNA区域进行高效转录,并且有助于提高引物特异性,提高全长DNA的得率(图4)。

High&#x20;yields&#x20;of&#x20;cDNA&#x20;over&#x20;a&#x20;broad&#x20;temperature&#x20;range

 

图4. 在广泛温度范围内的高cDNA得率。利用1 μg的Invitrogen Millennium RNA标记物(带有poly(A)-结构)和oligo(dT)18引物,使用Maxima H Minus反转录酶和来自其他供应商的反转录酶,按照说明书完成含有放射性标记cDNA合成,并进行对比。在碱性琼脂糖凝胶上分离反应产物。

长片断RT-PCR的扩增能力增强

Maxima H Minus反转录酶采用专利突变设计改善性能,能够从非常长的RNA模板合成全长cDNA(图5)。

Amplification&#x20;of&#x20;long&#x20;targets&#x20;in&#x20;two-step&#x20;RT-PCR

 

图5. 对两步法RT-PCR中长靶标的扩增。使用Maxima H Minus反转录酶和来自其他供应商的反转录酶,根据产品说明书以哺乳动物细胞的总RNA(1ug)进行重复反转录反应。将所得cDNA产物作为模板进行PCR扩增,并在琼脂糖凝胶上进行分离和观察。仅Maxima H Minus反转录酶能够生成非常长(13.3kb)的产物,并且具有高得率。

RT-qPCR的理想选择-- 灵敏度高和可重复性定量

Maxima反转录酶能够在较宽的模板量范围内完成可重复的cDNA合成,使其成为RT-qPCR实验的理想选择(图6)。Thermo Scientific Maxima第一链cDNA合成试剂盒中的预混液有助于进一步改善重复性,并节省反应组装过程的时间。

 

Reproducible&#x20;cDNA&#x20;synthesis&#x20;and&#x20;low&#x20;variability&#x20;with&#x20;a&#x20;wide&#x20;range&#x20;of&#x20;starting&#x20;RNA&#x20;amounts

图6. 在较宽的起始RNA量范围内,实现可重复的cDNA合成和低变异率(<1% SD/Ct)。在16个重复反应中,使用Maxima第一链cDNA合成试剂盒,以100、10、1、0.1、0.01和0.001 ng哺乳动物细胞总RNA合成第一链cDNA。以合成的cDNA为模板,使用Themo Scientific Maxima SYBR Green/ROX qPCR预混液进行qPCR扩增。扩增曲线图说明,在起始用量范围内具有良好的一致性。

具有完整的gDNA去除步骤,工作流程简单

Maxima第一链cDNA试剂盒包括双链特异性DNA酶(dsDNase),能够在cDNA合成前清除基因组DNA,无需任何中间样品纯化步骤。经过改造的dsDNase能够在2分钟内去除污染的基因组DNA,并且不会损害RNA数量或质量,也不会降解引物和探针等单链DNA(图7)。温和的热处理(55℃)即可轻松使dsDNase失活。所得无基因组DNA的RNA样品可直接用于cDNA合成反应,大大简化了实验工作流程。

Effective&#x20;removal&#x20;of&#x20;genomic&#x20;DNA&#x20;using&#x20;dsDNase

图7. 使用dsDNase有效去除基因组DNA。利用多种cDNA合成试剂盒,在有(RT +)和无(RT-)反转录酶的情况下对PBGD基因进行两步法RT-qPCR分析并绘图。使用具有dsDNase的Maxima第一链cDNA合成试剂盒或具有gDNA去除功能的其他供应商试剂盒,以0.2 ng总Jurkat RNA进行cDNA合成。使用Maxima试剂盒得到橙色、平坦的反转录反应曲线表明完全去除了污染的gDNA,而使用其他供应商试剂盒得到的反转录反应曲线显示具有残留gDNA的扩增。

在广泛的温度范围内能够有效保护RNA

RNA酶抑制剂通常会在高温条件下变性,释放RNA酶回到反应混合物中,随后损害和降解RNA。为了避免RNA被RNA酶破坏,所有Maxima第一链cDNA合成试剂盒均包含经改造的热稳定型Thermo Scientific RiboLock RNA酶抑制剂。蛋白质被“锁定”在RNA酶上,在高达55℃的温度下也能抑制RNA降解(图8)。RiboLock RNA酶抑制剂的高度热稳定性,对于在高反应温度下成功完成反转录至关重要。

RiboLock&#x20;RNase&#x20;Inhibitor&#x20;efficiently&#x20;protects&#x20;RNA&#x20;and&#x20;inhibits&#x20;up&#x20;to&#x20;2&#x20;ng&#x2f;20&#x20;&mu;L&#x20;of&#x20;RNase&#x20;A

图8. RiboLock RNA酶抑制剂可有效保护RNA并抑制高达2 ng/20 μL的RNA酶A。在37°C下,向人总RNA(1μg)分装样品(20 μL)中加入20 U的RiboLock RNA酶抑制剂,并提高RNase A的含量。
M:Thermo Scientific RiboRuler高分子量范围RNA Ladder,即用型
C:人总RNA
1:含RNAase A的人总RNA
2-5:含RiboLock RNA酶抑制剂和RNase A的人总RNA

RiboLock RNA酶抑制剂具有高度热稳定性。向人总RNA(1μg)分装样品(20 μL)中加入20 U的RiboLock RNA酶抑制剂和50 pg的RNase A,并在升高的温度下孵育。

 

了解分子进化

对聚合酶精细结构和功能的了解有限, 使我们使用合理的设计标准提高酶性能的想法受到了限制。通过模拟天然特性并使用定向进化来改善酶的性质,能够打破这种限制。

我们的专利技术——区室化核糖体展示(CRO)——能够实现快速、高效的反转录酶体外改进。该技术已经在野生型MMuLV反转录酶中引入和选择出多个有利突变,生成新型高度热稳定和高合成能力反转录酶,取代了相应的野生型反转录酶。

通过CRD技术进行分子改造包括几个步骤(图9)。首先,从野生型MMuLV反转录酶基因(1)开始,基于随机诱变创建mRNA文库(2)。接下来,在体外将mRNA文库翻译成与其mRNA前体相关的蛋白质(3)。然后,将蛋白质-mRNA复合物置于反转录酶反应混合物中并乳化,得到含有一个蛋白质-mRNA复合物的不同区室。最后,升高温度以产生选择性压力,在该选择压力下,只有改良突变体能够存活并产生全长cDNA(4)。通过结合性能最佳的突变,构建了高度进行性MMuLV RT突变体,其能够在高温下合成全长cDNA。

  1. Baranauskas A et al. (2012) Generation and characterization of new highly thermostable and processive M-MuLV reverse transcriptase variants. PEDS. doi:10.1093/protein/gzs034
  2. Skirgaila R et al. (2013) Compartmentalization of destabilized enzyme–mRNA–ribosome complexes generated by ribosome display: a novel tool for the directed evolution of enzymes. PEDS. doi:10.1093/protein/gzt017
     
Key&#x20;steps&#x20;in&#x20;the&#x20;molecular&#x20;evolution&#x20;of&#x20;MMuLV&#x20;RT

图9. MmuLV反转录酶分子改造的关键步骤

Comparison&#x20;of&#x20;RTs&#x20;and&#x20;cDNA&#x20;synthesis&#x20;with&#x20;or&#x20;without&#x20;functional&#x20;RNase&#x20;H&#x20;activity

Figure 10. Comparison of RTs and cDNA synthesis with or without functional RNase H activity.

Resources

Toools

Support

Contact us
Email or call our technical application scientists for additional questions regarding reverse transcription enzymes and master mixes.

OEM and custom solutions
Tailor-made performance tested products with strict quality standards to meet your molecular assay requirements.