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阿拉丁生产的EnzymoPure™III M-MLV反转录酶，即EnzymoPure™III M-MLV reverse transcriptase，是一种经过改造和优化的快速高效(短至10min完成反转录)、热稳定(高达55℃)、高精确度、产物超长(长达12kb)的反转录酶，具有正常的依赖于RNA或DNA模板的DNA聚合酶活性，能够以RNA或DNA为模板，在引物存在的情况下进行互补DNA链的合成，即可以进行cDNA(complementary DNA)的第一链合成。同时本反转录酶保留了RNase H活性，能选择性剪切RNA和DNA杂合双链中的RNA，有利于后续cDNA第二链的合成。本产品是最常用的优质反转录酶之一，广泛用于获得总RNA或mRNA后cDNA第一条链的合成，后续可以用于PCR、real-time PCR也称定量PCR(quantitative PCR, qPCR)、cDNA的第二链合成以及cDNA文库的构建，尤其是逆转录所得目的基因的克隆等。RTIIIM-MLV反转录酶还可以通过反转录用于DNA探针的荧光、生物素、地高辛或同位素标记等，也可以通过引物延伸(primer extension)来分析和研究RNA。M-MLV (Moloney Murine Leukemia Virus)，也称MMLV或M-MuLV；反转录酶(reverse transcriptase)也称逆转录酶或RT酶。本产品反转录快速高效，普通反转录仅需10min。实测小于3kb的片段，42℃反转录10min即可完成，与反转录60min相比无显著差别。大于3kb的片段，特别是大于6kb的片段，推荐反转录60min，以获得最佳的反转录效果。本产品反转录产物长度超长。本产品经测试可以轻松完成长度为8kb以下基因的反转录(参考图1)，反转录的最大长度可以达到12 kb。图1. 使用EnzymoPure™III M-MLV反转录酶反转录总RNA后，对于不同长度的cDNA进行PCR扩增后的电泳效果图。图中可见0.2-12kb的cDNA可以非常高效地被反转录。本产品热稳定性好，反转录效率高。本产品最适反应温度为42℃，当温度达到50℃时仍具有很高活性，对于长度较短的cDNA反转录，温度达到55℃时也可获得较高产量的cDNA。高温反转录对于高GC含量RNA的反转录，能有效减少二级结构，提升反转录效率。本产品反转录速度快，6kb以下的cDNA反转录只需0.5h即可完成，3kb以下的cDNA反转录10min即可完成(参考图2)。图2. EnzymoPure™III M-MLV反转录酶在不同温度反应不同时间的反转录效果。从HEK293T细胞抽提获得的总RNA 2μg，在20μl反转录体系中按照图中所示温度、时间进行反转录。反转录后取1μl反转录产物进行目的基因(2.6kb的YWHAZ基因和6.0kb的ADAR1基因)的PCR扩增和电泳。本产品性价比高。EnzymoPure™III M-MLV反转录酶是一种大肠杆菌重组高表达的反转录酶，由于其表达量非常高，大大提高了本产品的性价比。本反转录酶为表达含有从Moloney Murine Leukemia Virus reverse transcriptase基因克隆的pol基因，并进行了多个突变优化以提高其热稳定性、反转录效率、反转录长度和表达量。酶活性定义：One unit of the enzyme incorporates 1 nmol of dTTP into acid-precipitable material in 10 min at 37℃ using poly(A)oligo(dT)12-18 as template-primer。反应体系为 50 mM Tris-HCl(pH 8.3), 75 mM KCl, 3 mM MgCl2, 10 mM DTT, 0.5mM 3H]-dTTP and 0.4 mM polyAoligo(dT)12-18。纯度：不含DNA内切酶、外切酶和磷酸酯酶和RNA酶，可以满足常规反转录合成cDNA第一条链等的需要。酶储存溶液：20 mM Tris-HCl (pH 7.5), 100 mM NaCl, 0.1 mM EDTA, 1 mM DTT, 0.01%(v/v) NP-40 and 50%(v/v) glycerol。失活或抑制：80℃孵育10分钟可以导致EnzymoPure™III M-MLV反转录酶失活；EDTA、EGTA等螯合剂、无机磷酸盐或焦磷酸盐以及聚氨(polyamine)对EnzymoPure™III M-MLV反转录酶有抑制作用。本产品中M-MLV反转录酶的浓度为200U/μl，用于体积为20μl的反转录体系时，本试剂盒的不同包装足够分别进行50次、250次和1000次反转录反应。
酶储存溶液：
20 mM Tris-HCl (pH 7.5), 100 mM NaCl, 0.1 mM EDTA, 1 mM DTT, 0.01%(v/v) NP-40 and 50%(v/v) glycerol。失活或抑制：
80℃孵育10分钟可以导致III M-MLV反转录酶失活；EDTA、EGTA等螯合剂、无机磷酸盐或焦磷酸盐以及聚氨(polyamine)对III M-MLV反转录酶有抑制作用。本产品中M-MLV反转录酶的浓度为200U/μl，用于体积为20μl的反转录体系时，本试剂盒的不同包装足够分别进行50次、250次和1000次反转录反应。注意事项：
对于GC含量比较高的RNA的反转录，产品的使用说明中给予了特别说明，请予以关注。本产品仅限于专业人员的科学研究用，不得用于临床诊断或治疗，不得用于食品或药品，不得存放于普通住宅内。为了您的安全和健康，请穿实验服并戴一次性手套操作。使用说明：
1.cDNA第一条链的合成(First-stand cDNA Synthesis)：a.参考如下表格中设置的20μl反转录体系(RNase Inhibitor 和dNTP mix 可从阿拉丁订购)：模板(右侧3种任选其中一种)Total RNA0.1 ng-5 μg或poly(A) RNA/mRNA10 pg-0.5 μg或specific RNA0.01 pg-0.5 μg引物(右侧3种任选其中一种)Oligo(dT)18 primer0.5 μg(或100 pmol)或 Random Hexamer primer0.2 μg(或100 pmol)或 gene-specific primer15-25 pmolDEPC-treated Water－To 13.7μl*选择性步骤：如果模板RNA的GC含量较高(例如大于55%)或者有比较严重的二级结构，混匀后微离心以把液体沉降至管底，65℃孵育5min，随后立即置于冰上冷却，以打开RNA中一些比较稳定的二级结构。Reaction Buffer (5X)－4 μlRNase Inhibitor (40U/μl)－0.5 μldNTP Mix (25mM each)－0.8 μl**RT III M-MLV反转录酶－1 μl总体积20μl*To 13.7μl表示加入DEPC-treated Water至最终体积为13.7μl。**dNTP浓度不同时使用的体积需作适当调整，此时DEPC-treated Water的用量需适当调整。b.轻轻混匀(用移液器轻轻吹打混匀或用涡旋混合器在最低速度轻轻混匀)，随后离心沉淀液体。c.如果使用Oligo(dT)18或基因特异性引物，42℃孵育10-60 min。如果使用random hexamer(随机六聚体)作为引物，先在25℃孵育10 min，随后在42℃孵育10-60 min。反转录3kb以下的cDNA，反转录10min即可，反转录3-6kb的cDNA，反转录30min即可，而6kb以上的cDNA推荐反转录60min。当使用random hexamer(随机六聚体)作为引物，并且后续用于qPCR时，后续检测任何长度的基因，均反转录10min就足够了。注意：对于GC含量较高或二级结构比较严重的模板RNA，可以50℃孵育60min，以充分利用本产品在50℃时仍有良好的反转录酶活性这一特点，在较高温度进行反转录可以有效减少二级结构的干扰。d.80℃孵育10 min以失活RT III M-MLV反转录酶并终止反转录反应。说明：对于5kb以上的长片断cDNA不推荐采用加热的方法失活反转录酶，该方法易导致部分长片断DNA被剪切，此时可考虑酚氯仿抽提或柱纯化方法。e.反转录产物可以直接用于后续的PCR反应等，也可以-20℃冻存以备以后使用。用于后续PCR反应时，如果PCR的反应体系为20μl和50μl，则推荐相应地使用0.8μl和2μl反转录产物。2.引物延伸、探针标记等其它用途请自行参考M-MLV反转录酶的相关文献资料进行。常见问题：1.总RNA反转录产物电泳观察不到。a.反转录产物由于是从模板反转录而获得，而模板的量本身比较低，反转录的量通常还要少于模板量，并且总RNA的反转录产物大小很不均匀，因此通常总RNA的反转录产物直接电泳观察是观察不到的。 2.反转录产物通过PCR扩增没有特异性条带。a.PCR扩增没有获得特异性条带时建议先使用actin、GAPDH等作为内参进行PCR扩增，看是否可以成功扩增。如果可以成功，则说明PCR扩增体系没有问题，此时通常是目的基因的引物设计欠佳，当然也有可能是反转录产物质量欠佳。如果内参不能被很好地扩增，则有可能PCR体系存在问题或反转录产物质量欠佳。b.模板RNA发生了降解。哺乳动物细胞或组织的总RNA琼脂糖电泳后应该可以看到清晰的18S和28S rRNA条带，并且28S rRNA和18S rRNA的亮度比例应该大于等于2.0。如果比例小于2.0，则提示总RNA发生了显著的降解，最好能重新制备总RNA样品。避免RNA降解的主要方法是，严格进行RNA的相关操作，包括带洁净手套、戴一次性口罩、在洁净环境中抽提或制备RNA，以尽量避免RNase污染。c.模板RNA的纯度偏低。在提取纯化RNA的过程中，残留在溶液中的一些成分如苯酚、SDS、EDTA、胍盐、磷酸、焦磷酸、多胺、亚精胺等会抑制反转录酶活性。对RNA样品进行柱纯化，或者进行沉淀、洗涤和再溶解，通常可以有效去除残留的污染物。通常选择使用阿拉丁的Zol或Trizol抽提获得的总RNA完全可以满足反转录反应的需要。d.反转录反应的模板量不足。在抽提获得总RNA后，在进行一些精细的定量检测时通常会进行DNase I消化，以充分去除可能的残留的DNA的干扰。DNase I进行热失活时，需要加入EDTA至终浓度为2.5mM，否则RNA在没有螯合剂的情况下，在加热过程中容易被水解，从而导致模板量不足。此外，扩增特定基因时，需要先查询该基因的组织分布特点，利用其高表达的组织进行目的基因的反转录和克隆。用该基因丰度极低的组织或细胞样品进行反转录和PCR扩增，通常会由于模板量过少而PCR扩增失败。e.没有使用适当的反转录引物。对于细菌RNA和不含poly(A)尾巴的RNA，要用random hexamer引物代替Oligo(dT)18引物。使用基因特异性反转录引物时，需要确保基因特异性引物设计合理正确。f.如果RNA模板富含GC或容易形成二级结构，此时可以考虑把反转录温度提高到45-55℃。EnzymoPure™III M-MLV reverse transcriptase is a DNA polymerase that uses single stranded RNA or DNA as template to synthesize the first-strand complementary DNA strands (cDNA) rapidly and efficiently in the presence of primers. It is optimized to have a high thermal stability and high fidelity, and can synthesize long cDNA up to 12kb. Additionally, this product has RNase H activity that selectively degrades the RNA strand of an RNA-DNA hybrid, thus facilitating the synthesis of second-strand cDNA subsequently.EnzymoPure™III M-MLV Reverse Transcriptase is one of the most widely used high-quality reverse transcriptase. It is mainly used for synthesizing first-strand cDNA with total RNA or mRNA template. The obtained cDNA can be used directly for PCR, real-time PCR, second-strand cDNA synthesis and construction of cDNA libraries, etc. Moreover, it can also be used to perform RNA analysis by primer extension, or label DNA probes with fluorescence, biotin, digoxin or isotope.EnzymoPure™III M-MLV Reverse Transcriptase is a high efficient reverse transcriptase, requiring 10 minutes only to synthesize cDNA no longer than 3kb. To synthesize cDNA longer than 3kb, especially longer than 6kb, 60min of reverse transcription is recommended.EnzymoPure™III M-MLV Reverse Transcriptase is a high efficient reverse transcriptase, requiring 10 minutes only to synthesize cDNA no longer than 3kb. To synthesize cDNA longer than 3kb, especially longer than 6kb, 60min of reverse transcription is recommended.This product is highly cost-effective. EnzymoPure™III M-MLV Reverse Transcriptase is a recombinant reverse transcriptase expressed and purified from E. coli transformed with the expression plasmids carrying the pol gene of M-MLV with RNase H coding region included. The enzyme has been engineered to have better thermal stability and higher reverse transcription efficiency, and produce longer reverse transcript.Definition of enzyme activity
Inactivation or inhibition：
RT III M-MLV Reverse Transcriptase can be inactivated by incubation at 80ºC for 10 minutes, or inhibited by EDTA, EGTA , inorganic phosphates, pyrophosphates and polyamine.This product is able to synthesize long cDNA. It enables easy reverse transcription of genes less than 8kb (Figure 1). The maximum length of cDNA can be up to 12kb. Figure 1. Agarose gel electrophoresis of PCR products amplified from the cDNA template synthesized with the RT III M-MLV Reverse Transcriptase (, #D7176). cDNA ranging from 0.2kb to 12kb in length can be synthesized high efficiently.RT III M-MLV Reverse Transcriptase has good thermal stability and high reverse transcription efficiency. It has an optimum working temperature of 42℃, but it is still highly active at 50℃. Reverse transcription at higher temperature can significantly improve the reverse transcription of RNA with high GC content and complex secondary structures. This reverse transcriptase has a high reverse transcription rate, requiring 30 minutes only to complete the synthesis of cDNA less than 6kb, and 10min only to obtain cDNA less than 3kb (Figure 2). Figure 2. Agarose gel electrophoresis of PCR products amplified from the cDNA template synthesized by the RT III M-MLV Reverse Transcriptase (, #D7176). 2µg total RNA extracted from HEK293T cells was reverse transcribed in a reaction volume of 20µl at different temperatures for different duration of reverse transcription, as indicated in the figure. After reverse transcription, 1µl reverse transcription product was used to amplify YWHAZ and ADAR1 genes of 2.6kb and 6.0kb, respectively.The concentration of RT III M-MLV Reverse Transcriptase is 200U/µl. When 20µl of reverse transcription volume is used, the D7176S, D7176M and D7176L are sufficient for 50, 250 and 1000 reactions, respectively.Precautions：
Please refer to the instructions for reverse transcription of RNA with high GC content.This product is for R&D only. Not for drug, household, or other uses.For your safety and health, please wear lab coat and disposable gloves during the operation.Instructions for Use：
1. First strand cDNA synthesis.a. Set up the first-strand cDNA synthesis in a nuclease free PCR tube on ice or at room temperature as follows. RNase Inhibitor and dNTP mix can be purchased from .Template (one of the three types of RNA)Total RNA0.1ng-5μgPoly(A) RNA/mRNA10pg-0.5μgSpecific RNA0.01pg-0.5μgPrimer (one of the three types of primer)Oligo(dT)18 Primer0.5μg (or 100pmol)Random Hexamer Primer0.2μg (or 100pmol)Gene-specific Primer15-25pmolDEPC-treated Water-To 13.7μl*(Optional) For RNAs with high GC content (e.g. ＞55%) or complex secondary structures, incubate the mixture of primer and template at 65ºC for 5 minutes, and immediately put it on ice to disrupt RNA secondary structures.Reaction Buffer (5X)-4μlRNase Inhibitor (40U/μl)-0.5μldNTP Mix (25mM each)-0.8μl**RT III M-MLV Reverse Transcriptase-1μlTotal Volume-To 20μl* ‘To 13.7μl’ means filling the mixture of template and primer to a total volume of 13.7μl with DEPC-treated water.** The volume of dNTP mix varies depending on the concentration of dNTP stock. If the volume of dNTP is not 0.8µl, adjust with DEPC-treated water accordingly.b. Mix the reaction by vortex or pipetting gently, centrifuge briefly to allow liquid to accumulate at the bottom of PCR tube.c. Incubate the reaction at 42℃for 10-60min if Oligo(dT)18 primer or gene-specific primer is used. If random hexamer is used, incubate at 25℃for 10min, followed by incubation at 42℃for 10-60min. For cDNA less than 3kb, 10-minute incubation is usually sufficient. For cDNA between 3kb and 6kb, we recommend 30 minutes incubation. While for cDNA exceeding 6kb in length, 60-minute incubation is recommended. When using random hexamers for the reverse transcription which will be followed by qPCR experiments, 10-minute incubation is sufficient regardless of the length of cDNA.Note: For RNA template with high GC content or secondary structures, incubate the reaction at 50℃for 60min. The RT III M-MLV Reverse Transcriptase maintains good reverse transcriptase activity at 50ºC, and reverse transcription at a higher temperature effectively reduce the interference of secondary structures.d. Stop the reverse transcription by incubating the reaction at 80℃for 10min to inactivate RT III M-MLV Reverse Transcriptase. Note: Heat-inactivation of reverse transcriptase is not recommended for long cDNA over 5kb, as this method may cause shearing of long cDNA fragments. e. The reverse transcription products can be used directly for subsequent experiments such as PCR, or stored at -20℃for future use. We recommend using 0.8μl and 2μl reverse transcription products in a PCR reaction volume of 20μl and 50μl, respectively.2. For other applications such as primer extension and probe labeling, please refer to reference related to M-MuLV reverse transcriptase (RNase H-).FAQ:1. The reverse transcription product of total RNA is invisible after electrophoresis.It is a normal phenomena, because the amount of RNA template is low, and the amount of reverse transcription products in different size is even lower. 2. No specific product can be amplified from the reverse transcription product.a. To exclude the problem of PCR reaction system or reverse transcription product, use gene-specific primers to amplify internal reference genes, such as actin and GAPDH. If reference genes can be amplified, but not the target gene, it indicates primers of target gene are not well designed, the expression of the target gene is too low to be detected, or the reverse transcription efficiency is low. b. Template RNA may be degraded. The integrity of total RNA can be checked by agarose gel or on-chip electrophoresis. Intact total RNA exhibits sharp, clear 28S and 18S rRNA bands, and the 28S rRNA band should be approximately twice as intense as the the 18S rRNA band. A ratio less than 2 indicates the degradation of total RNA and new total RNA should be prepared. c. RNA samples may contain some components that inhibit the activity of reverse transcriptase. Those contaminants include phenol, SDS, EDTA, guanidine salts, phosphoric acid, pyrophosphoric acid, polyamine, and spermidine etc, which can be removed effectively by column purification or precipitation, washing, and redissolution. Total RNA extracted by Zol (, R0011) or Trizol (, R0016) usually can meet the requirements of reverse transcription.d. Insufficient templates for reverse transcription. When DNase I used to remove the residual DNA in RNA sample is subjected to heat-inactivation prior to reverse transcription, EDTA should be added to RNA sample at a final concentration of 2.5mM to protect RNA from degradation under high temperature. Additionally, to amplify a specific gene, it is necessary to extract RNA from tissues in which the target gene is highly expressed. e. Inappropriate primer is used for reverse transcription. Random hexamer instead of Oligo(dT)18 should be used for the reverse transcription of bacterial total RNA which does not have poly(A) tails. Gene-specific primers used for reverse transcription must be well designed.f. For RNA template with high GC content or secondary structures, increase the reverse transcription temperature to 45-50℃ to improve the reverse transcription efficiency.
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