QuantiTect Reverse Transcription Kit
For fast cDNA synthesis enabling sensitive real-time two-step RT-PCR for gene expression analysis
- cDNA synthesis and gDNA removal in only 20 minutes
- High cDNA yields even from low-abundance transcripts
- cDNA synthesis from 5' and 3' regions of transcripts
- No need to design RNA-specific primers or probes
The unique QuantiTect Reverse Transcription Kit provides a fast and convenient procedure for cDNA synthesis with integrated genomic DNA removal. Genomic DNA contamination in RNA samples is effectively eliminated by gDNA Wipeout Buffer. All components that are required for fast and efficient reverse transcription are provided with the QuantiTect Reverse Transcription Kit, including Quantiscript Reverse Transcriptase, Quantiscript RT Buffer, and a unique RT Primer Mix. The synthesized cDNA is optimized for use in real-time PCR, allowing reliable quantification of targets from all regions of mRNA transcripts.
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QuantiTect Rev. Transcription Kit (50)
For 50 x 20 µl reactions: 100 µl 7x gDNA Wipeout Buffer, 50 µl Quantiscript Reverse Transcriptase, 200 µl 5x Quantiscript RT Buffer, 50 µl RT Primer Mix, 1.9 ml RNase-Free Water
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205311
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QuantiTect Rev. Transcription Kit (200)
For 200 x 20 µl reactions: 4 x 100 µl 7x gDNA Wipeout Buffer, 4 x 50 µl Quantiscript Reverse Transcriptase, 4 x 200 µl 5x Quantiscript RT Buffer, 4 x 50 µl RT Primer Mix, 4 x 1.9 ml RNase-Free Water
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205313
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QuantiTect Rev. Transcription Kit (400)
For 400 x 20 µl reactions: 800 µl 7x gDNA Wipeout Buffer, 400 µl Quantiscript Reverse Transcriptase, 1.6 ml 5x Quantiscript RT Buffer, 400 µl RT Primer Mix, 8 x 1.9 ml RNase-Free Water
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205314
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QuantiTect Reverse Transcription Kit适用于分子生物学应用。该产品不适用于疾病的诊断、预防或治疗。
快速、便利的cDNA合成。|高效去除基因组DNA,获得准确的real-time RT-PCR。|灵敏检测5'端12.5 kb的转录本。|两步法real-time RT-PCR具有高灵敏性。|
使用QuantiTect Reverse Transcription Kit,只需20分钟即可完成基因组DNA去除和cDNA合成。由于上述反应均采用相同的孵育温度且使用预混液构建,因此操作步骤十分快速便捷。而Supplier I的试剂盒的操作步骤则长很多,它具有其他移液步骤且频繁改变孵育温度,因此需要更多的“操作时间”。|使用 (+RT) 或不使用 (–RT) 逆转录酶,对β-actin进行两步法real-time RT-PCR。利用100 ng总RNA合成cDNA,在LightCycler 2.0上使用QuantiTect SYBR® Green PCR Kit进行real-time PCR,重复两次。利用β-actin特异性引物检测mRNA和基因组DNA序列。同时进行无模板对照反应 (绿色)。[A] 使用QuantiTect Reverse Transcription Kit进行RT步骤。红色的平直–RT曲线表示有效去除了残留基因组DNA。[B] 使用Supplier I (Enzyme SIII) 的试剂盒进行逆转录步骤。紫色的–RT曲线表示残留基因组DNA扩增。|位于小鼠抗肌萎缩蛋白基因5'区域的靶点(poly-A位点上游约12.5 kb)的两步法real-time RT-PCR。使用QuantiTect Reverse Transcription Kit及Supplier I和Supplier R提供的逆转录酶,对从小鼠睾丸中纯化的总RNA进行逆转录。在LightCycler系统上采用real-time PCR分析相同体积的三次重复的逆转录反应。误差条显示了三次重复反应的标准差。与其他两个试剂盒相比,QuantiTect Reverse Transcription Kit可生成更多的cDNA(表现为较低的CT值),并且real-time RT-PCR的可重复性更高(表现为更小的误差范围)。RFU:相对荧光单位。
(数据由德国汉堡大学医院分子男科学系的Andrej-Nikolai Spiess博士提供。)|[A] TGFB2(低表达)和 [B] IL8(高表达)的两步法real-time RT-PCR分析。使用PAXgene Blood RNA系统从人全血中纯化总RNA。然后使用QuantiTect Reverse Transcription Kit、Supplier AII的试剂盒或Supplier I的试剂盒从1 µg RNA中合成cDNA。在ABI PRISM 7900上使用QuantiTect Probe PCR Kit及针对TGFB2或IL8的QuantiTect Gene Expression Assay进行real-time PCR,重复两次。使用QuantiTect Reverse Transcription Kit扩增TGFB2,获得最低的CT值,证明即便是低丰度的基因亦可高效逆转录,并在real-time PCR中实现灵敏的检测。|
性能
Using the QuantiTect Reverse Transcription Kit, contaminating genomic DNA in RNA samples is effectively and rapidly removed with the unique gDNA Wipeout Buffer (see figure Effective genomic DNA removal for accurate real-time RT-PCR). Elimination of genomic DNA is crucial for accurate gene expression results, and design of RNA-specific primers or probes is not always possible. With gDNA Wipeout Buffer, time is saved and costs are reduced, since a separate DNase digestion is unnecessary, either during or after purification of RNA samples.
The high RNA affinity of Quantiscript Reverse Transcriptase, in combination with Quantiscript RT Buffer, enables high yields of cDNA from any RNA template (see table “Higher cDNA yields for less abundant transcripts”). Even difficult templates, such as those with high GC-content or complex secondary structure, are successfully reverse transcribed.
| 1000 |
30.9 |
32.0 |
23.1 |
24.9 |
| 100 |
34.2 |
35.4 |
26.3 |
26.6 |
| 10 |
37.8 |
46.8 |
29.7 |
30.3 |
| 1 |
N.D. |
N.D. |
32.4 |
34.5 |
The RT Primer Mix contains a specially optimized mix of oligo-dT and random primers that enable cDNA synthesis from all regions of RNA transcripts, even from 5' regions (see figure Sensitive detection of a target at the 5' region of a 12.5 kb transcript). In contrast to kits from other suppliers, the QuantiTect Reverse Transcription Kit provides high yields of cDNA template for real-time PCR analysis regardless of where the amplified target region is located on the transcript, and provides greater sensitivity in the detection of low-abundance genes (see figure "Higher sensitivity in real-time, two-step RT-PCR"). The QuantiTect Reverse Transcription Kit also enables greater reproducibility in real-time RT-PCR.
原理
QuantiTect Reverse Transcriptase is a novel blend of Omniscript and Sensiscript Reverse Transcriptases, which has a high affinity for RNA and is capable of cDNA synthesis from a wide range of RNA amounts (10 pg to 1 µg). In contrast to kits from other suppliers, the QuantiTect Reverse Transcription Kit provides high yields of cDNA template for real-time PCR analysis regardless of where the amplified target region is located on the transcript. Even difficult templates, such as those with high GC-content or complex secondary structure, are successfully reverse transcribed. QuantiTect RT Buffer has also been optimized to be compatible with real-time PCR buffer.
To obtain accurate results in real-time RT-PCR gene expression assays, it is important that only cDNA is amplified and detected. Interference by genomic DNA can be avoided by designing primers or probes that span an exon/exon boundary. However, in cases where this is not possible (e.g., the cDNA is from a single-exon gene), it is essential that the starting RNA sample is free of genomic DNA. Using the QuantiTect Reverse Transcription Kit, contaminating genomic DNA in RNA samples is effectively and rapidly removed with unique gDNA Wipeout Buffer. Time is saved and costs are reduced, since a separate DNase digestion not required, either during or after purification of RNA samples. Also, design of RNA-specific primers or probes is unnecessary.
| gDNA Wipeout Buffer |
Detection of RNA only in real-time RT-PCR |
| Quantiscript Reverse Transcriptase |
Use of a wide range of RNA amounts (10 pg to 1 µg RNA) High sensitivity |
| Quantiscript RT Buffer |
Read-through of difficult templates |
| RT Primer Mix |
cDNA synthesis from all regions of transcripts, even from 5' regions |
操作流程
Genomic DNA removal and cDNA synthesis take only 20 minutes with the QuantiTect Reverse Transcription Kit (see flowchart "Fast and convenient cDNA synthesis"). The procedure is fast and convenient, since both reactions are run using the same incubation temperature and are set up using master mixes.
The QuantiTect Reverse Transcription Kit includes everything you need for fast cDNA synthesis. Purified RNA is briefly incubated in gDNA Wipeout Buffer to effectively remove contaminating genomic DNA. In contrast to other methods, the RNA sample is then used directly in reverse transcription, using a master mix prepared from Quantiscript Reverse Transcriptase, Quantiscript RT Buffer, and RT Primer Mix. With Quantiscript Reverse Transcriptase, RNA can be transcribed at low temperatures, even through complex 2° structure, ensuring that the RNA will stay intact — the entire reaction takes place at 42°C and is then inactivated at 95°C. Additional steps for RNA denaturation, primer annealing, and RNase H digestion are not necessary.
应用
The QuantiTect Reverse Transcription Kit allows highly efficient and sensitive real-time RT-PCR for all types of starting material, including laser-microdissected samples and tissue biopsies.
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特点
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参数
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应用
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Quantification of (even low-abundance) transcripts
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酶活
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Reverse transcription
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预混液
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No
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反应类型
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Two-step, cDNA production, genomic DNA digestion
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real-time或终点法PCR
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Real time
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样本/目标类型
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RNA template
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单一或多重
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Single
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FAQ ID -111
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For cDNA synthesis with integrated removal of genomic DNA contamination
For use in real-time two-step RT-PCR
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快速、便利的cDNA合成。
使用QuantiTect Reverse Transcription Kit,只需20分钟即可完成基因组DNA去除和cDNA合成。由于上述反应均采用相同的孵育温度且使用预混液构建,因此操作步骤十分快速便捷。而Supplier I的试剂盒的操作步骤则长很多,它具有其他移液步骤且频繁改变孵育温度,因此需要更多的“操作时间”。
高效去除基因组DNA,获得准确的real-time RT-PCR。
使用 (+RT) 或不使用 (–RT) 逆转录酶,对β-actin进行两步法real-time RT-PCR。利用100 ng总RNA合成cDNA,在LightCycler 2.0上使用QuantiTect SYBR® Green PCR Kit进行real-time PCR,重复两次。利用β-actin特异性引物检测mRNA和基因组DNA序列。同时进行无模板对照反应 (绿色)。[A] 使用QuantiTect Reverse Transcription Kit进行RT步骤。红色的平直–RT曲线表示有效去除了残留基因组DNA。[B] 使用Supplier I (Enzyme SIII) 的试剂盒进行逆转录步骤。紫色的–RT曲线表示残留基因组DNA扩增。
灵敏检测5'端12.5 kb的转录本。
位于小鼠抗肌萎缩蛋白基因5'区域的靶点(poly-A位点上游约12.5 kb)的两步法real-time RT-PCR。使用QuantiTect Reverse Transcription Kit及Supplier I和Supplier R提供的逆转录酶,对从小鼠睾丸中纯化的总RNA进行逆转录。在LightCycler系统上采用real-time PCR分析相同体积的三次重复的逆转录反应。误差条显示了三次重复反应的标准差。与其他两个试剂盒相比,QuantiTect Reverse Transcription Kit可生成更多的cDNA(表现为较低的CT值),并且real-time RT-PCR的可重复性更高(表现为更小的误差范围)。RFU:相对荧光单位。
(数据由德国汉堡大学医院分子男科学系的Andrej-Nikolai Spiess博士提供。)
两步法real-time RT-PCR具有高灵敏性。
[A] TGFB2(低表达)和 [B] IL8(高表达)的两步法real-time RT-PCR分析。使用PAXgene Blood RNA系统从人全血中纯化总RNA。然后使用QuantiTect Reverse Transcription Kit、Supplier AII的试剂盒或Supplier I的试剂盒从1 µg RNA中合成cDNA。在ABI PRISM 7900上使用QuantiTect Probe PCR Kit及针对TGFB2或IL8的QuantiTect Gene Expression Assay进行real-time PCR,重复两次。使用QuantiTect Reverse Transcription Kit扩增TGFB2,获得最低的CT值,证明即便是低丰度的基因亦可高效逆转录,并在real-time PCR中实现灵敏的检测。
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