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NGS Library Prep kits

Molecular Research

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HiScript II Reverse Transcriptase

HiScript® II Reverse Transcriptase is a new reverse transcriptase obtained through in vitro molecular evolution technology on the basis of M-MLV (RNase H-) Reverse Transcriptase. Compared with the previous generation HiScript® Reverse Transcriptase, HiScript® II Transcriptase has further greatly improved thermal stability, with a half-life of more than 4 hours at 50°C, and can be reacted for a long time at 55°C to maintain stability. It is very suitable for complex secondary Reverse transcription of structural RNA template. In addition, HiScript® II Transcriptase adds a number of point mutations, which further enhances template affinity and progression, greatly improving the synthesis ability of full-length cDNA, and obtaining cDNA up to 20 kb. It has a higher tolerance to common reverse transcription inhibitors and is very suitable for the reverse transcription reaction of plant tissue RNA rich in polysaccharides and polyphenols.   The half-life at 50℃ exceeds 240 min, and the half-life at 55℃ can reach 60 min; CDNA up to 10 kb can be synthesized in 5 minutes, which is suitable for rapid reverse transcription reaction; Higher impurity tolerance and higher detection sensitivity, reverse transcriptase is the first choice for molecular diagnosis   Store at -20℃
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R201-01/02
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HiScript® II Reverse Transcriptase is a new reverse transcriptase obtained through in vitro molecular evolution technology on the basis of M-MLV (RNase H-) Reverse Transcriptase. Compared with the previous generation HiScript® Reverse Transcriptase, HiScript® II Transcriptase has further greatly improved thermal stability, with a half-life of more than 4 hours at 50°C, and can be reacted for a long time at 55°C to maintain stability. It is very suitable for complex secondary Reverse transcription of structural RNA template. In addition, HiScript® II Transcriptase adds a number of point mutations, which further enhances template affinity and progression, greatly improving the synthesis ability of full-length cDNA, and obtaining cDNA up to 20 kb. It has a higher tolerance to common reverse transcription inhibitors and is very suitable for the reverse transcription reaction of plant tissue RNA rich in polysaccharides and polyphenols.

 

The half-life at 50℃ exceeds 240 min, and the half-life at 55℃ can reach 60 min;
CDNA up to 10 kb can be synthesized in 5 minutes, which is suitable for rapid reverse transcription reaction;
Higher impurity tolerance and higher detection sensitivity, reverse transcriptase is the first choice for molecular diagnosis

 

Store at -20℃

Keyword:
R201-01/02
We could not find any corresponding parameters, please add them to the properties table

Q1:原核生物RNA能用我们的逆转录试剂吗?

A1:可以。原核生物RNA没有polyA尾,所以在逆转录反应体系中需要以Random primer作为逆转录引物。

Q2:逆转录试剂盒中带有gDNA wiper Mix,可以去除残留在RNA中的基因组。最多可以去除多少的基因组?但如果不进行基因组去除,会影响接下来的逆转录吗?

A2:我们做过100ng基因组的测试,去除效率是99.95%。如果不进行基因组去除不会影响逆转录实验的进行,但是如果基因组残留严重但不去除的话,会影响接下来定量实验的准确性。

Q3:延长逆转录时间,是否可以提高逆转录效率?

A3:对于大多数基因,延长逆转录时间对逆转录效率并没有显著提升;对于一些GC含量较高或含高级结构的模板,延长逆转录时间可提升逆转录效率。

Q4:做了No RT Control发现有gDNA残留怎么办?

A4:如果使用试剂盒中的gDNA wiper模块进行gDNA去除,但No RT Control仍有CT值,可通过实验组和No RT Control组的CT值来判断实验数据是否可用。当实验组CT值与No RT Control对照组CT值差值大于5,说明由gDNA导致的误差小于5%,则可以忽略gDNA污染;若实验组与对照组CT值差值小于3或者几乎一致,则实验组扩增产物的模板很大一部分来源于gDNA,这样的实验组就失去定量意义。

Q5:如何评判RNA质量?

A5:RNA质量从两个方面反应:

(1) RNA完整度。通过琼脂糖凝胶电泳验证RNA完整度。以真核生物为例,完整的总RNA有清晰的三条带,分子量从大到小分别为28S、18S、5S,并且28S是18S亮度的两倍;若能看见三条带,但带型模糊或弥散,则说明RNA有部分降解,此时请立即进行逆转录反应,并适量加大模板量;若只能看见分子量很小的一条带或没有条带,则 RNA 已完全降解,需要重新提取。

(2) RNA的纯度。RNA的纯度可以通过OD260/280和OD260/230两个比值是否在1.8-2.1范围内进行判断,蛋白、离子等残留会使比值降低。

Q6:如何选择逆转录的引物?

A6:根据实验的具体情况选择随机引物、Oligo dT或基因特异性引物进行逆转录。

(1) 随机引物:随机结合在RNA的任何区域,适用于rRNA、mRNA、tRNA等所有RNA类型的逆转录反应。

(2) Oligo dT:适用于具有PolyA尾的RNA(原核生物RNA、真核生物的rRNA和tRNA不具有PolyA尾)。 由于Oligo dT结合在PolyA尾上,所以对 RNA 样品的质量要求较高,即使有少量的降解也会使全长cDNA合成量大大减少。

(3) 基因特异性引物:与模板序列互补,适用于序列已知的基因逆转录。

cDNA后续进行PCR扩增长片段,一般用Oligo dT或者基因特异性引物,不建议使用随机引物,因为随机引物是随机结合的,cDNA片段会偏短,可能会对长片段扩增造成稀释,后续扩增不出全长的目的基因;cDNA后续进行qPCR,一般使用Oligo dT和随机引物的混合物,可以避免3’端和5’端的扩增偏好性。

Q7:可以通过测逆转产物cDNA浓度判定逆转效率吗?

A7:不可以,逆转录产物cDNA是混合物,除了cDNA产物以外,还有buffer、逆转录酶、引物,同时还包含未转录的模板RNA,总RNA中的tRNA、rRNA等,都会干扰浓度测定结果,因此不能反应真实的cDNA产量。

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