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阿拉丁生产的EnzymoPure™Q M-MLV反转录酶，即EnzymoPure™Q M-MLV reverse transcriptase，是一种经过改造和优化特别适合定量PCR(quantitative PCR, qPCR)的高精确度反转录酶。EnzymoPure™Q M-MLV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。同时本反转录酶还保留了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，特别适合用于qPCR和一步法的qRT-PCR，本产品的反转录产物也适合用于常规的PCR、cDNA的第二链合成以及cDNA文库的构建，以及逆转录所得目的基因的克隆等。EnzymoPure™Q M-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。本产品反转录性能优、检测灵敏度高、扩增特异性强、反应稳定性好，不仅非常适合用于常规的RNA样品的反转录和qPCR检测或一步法qRT-PCR检测，也非常适合于內源低丰度RNA、外源病毒RNA等微量RNA的反转录及后续的qPCR检测。利用EnzymoPure™Q M-MLV反转录酶对总RNA进行反转录，之后以不同稀释倍数的cDNA为模板，通过qPCR检测目的基因的结果请参考图1。图1.EnzymoPure™Q M-MLV反转录酶对总RNA进行反转录后用于qPCR检测的效果图。从HEK293T细胞抽提获得的总RNA 2µg，在20μl反转录体系(2µg Total RNA, 0.5μg Oligo(dT)18 primer, 0.2μg Random Hexamer primer, 1X Reaction Buffer, 20U RNase Inhibitor, dNTP Mix (1mM each), 200U EnzymoPure™Q M-MLV反转录酶)中进行反转录，反转录后取1μl (100%)或不同稀释比例(1μl的10%、1%、0.1%、0.01%)反转录产物进行GAPDH cDNA 151bp目的片段的qPCR扩增和电泳检测。A.扩增曲线图。B.溶解曲线图。C.qPCR扩增产物的电泳图。NTC (no-template control)，用水代替反转录产物。qPCR反应体系20µl，使用阿拉丁生产的D7260 UltraBio™ SYBR Green qPCR Mix (2X)。实际检测效果会和特定实验条件有关，图中结果仅供参考。
酶储存溶液：
20mM Tris-HCl (pH7.5), 150mM NaCl, 0.1mM EDTA, 1mM 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RT Q 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RT Q 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。关于阿拉丁不同反转录酶的比较和选择，可参考相关网页：http://www.aladdin-e.com/support/reversetranscriptase.htm'],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 RNA0.1ng-5μg或poly(A) RNA/mRNA10pg-0.5μg或specific RNA0.01pg-0.5μg引物(右侧3种任选其中一种)Oligo(dT)18 primer0.5μg(或100pmol)或Random Hexamer primer0.2μg(或100pmol)或gene-specific primer15-25pmolDEPC-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)-4μlRNase Inhibitor (40U/μl)-0.5μldNTP Mix (25mM each)-0.8μl**RT QM-MLV反转录酶-1μl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10-60min。如果使用random hexamer(随机六聚体)作为引物，先在25℃孵育10min，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。d.80℃孵育10 min以失活RT Q M-MLV反转录酶并终止反转录反应。注：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.qPCR检测。a.推荐使用阿拉丁生产的D7260 SYBR Green qPCR Mix (2X)、D7262 SYBR Green qPCR Mix (2X, Low ROX)、D7265 SYBR Green qPCR Mix (2X, High ROX)进行常规的荧光染料法qPCR检测，或者使用阿拉丁生产的D7271 Probe qPCR Mix (2X)、D7272 Probe qPCR Mix (2X, Low ROX)、D7273 Probe qPCR Mix (2X, High ROX)进行Taqman探针法qPCR检测。b.qPCR的具体条件，请参考qPCR试剂盒的相关说明。3.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。EnzymoPure™Q M-MLV Reverse Transcriptase uses single-stranded RNA or DNA in the presence of a primer to synthesize a complementary DNA strand with high fidelity, sensitivity, and specificity. It is a modified, optimized reverse transcriptase particularly suitable for qPCR analysis. The EnzymoPure™Q M-MLV Reverse Transcriptase contains RNase H enzyme activity that can specifically degrade the RNA strand of an RNA-DNA hybrid, enabling the subsequent synthesis of second-strand cDNA.EnzymoPure™Q M-MLV Reverse Transcriptase, a commonly used high-quality reverse transcriptase, is widely used in the synthesis of first-strand cDNA using total RNA or mRNA templates, and is particularly suitable for qPCR and one-step qRT-PCR analysis. This product is also applicable for conventional PCR, synthesis of second-strand cDNA and construction of cDNA library, and cloning of target genes. It can also be used for DNA probe labeling with fluorescence, biotin, digoxin, or isotope through reverse transcription reaction, or for RNA studies by primer extension.Please refer to Figure 1 for the qPCR analysis of the GADPH gene in HEK293T cells using the cDNA templates obtained by the EnzymoPure™Q M-MLV reverse transcriptase.Figure 1
Source：
Recombinant reverse transcriptase expressed in E. coli.Definition of enzyme activity unit: One unit is defined as the amount of enzyme that incorporates 1 nmol of dTTP into acid-precipitable material in 10min at 37℃ using poly(A)•oligo(dT)12-18 as template-primer. Reaction system: 50mM Tris-HCl (pH8.3), 75mM KCl, 3mM MgCl2, 10mM DTT, 0.5mM [3H]-dTTP, and 0.4mM polyA•oligo(dT)12-18.Purity: This product is free from DNA endonuclease, exonuclease, phosphatase, and RNase, and can meet the requirements of conventional reverse transcription.Storage buffer: 20mM Tris-HCl (pH7.5), 150mM NaCl, 0.1mM EDTA, 1mM DTT, 0.01%(v/v) NP-40, and 50%(v/v) glycerolInactivation or inhibition：
RT Q M-MLV Reverse Transcriptase can be inactivated by incubation at 80℃ for 10 minutes, or inhibited by EDTA and EGTA chelators, inorganic phosphates or pyrophosphates, and polyamine.The concentration of M-MLV is 200U/µl. When 20µl of reverse transcription reaction volume is used, D7188S, D7188M, and D7188L are sufficient for 50, 250, and 1000 reactions, respectively. provides a variety of reverse transcriptase. To select the best one for your needs, please refer to the following webpage:http://www.aladdin-e.com/support/reversetranscriptase.htmPrecautions：
Please refer to the instructions for reverse transcription of RNAs 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 a lab coat and disposable gloves during the operation.Instructions for Use：
1. First-strand cDNA synthesisa. Set up the following reaction on ice or at room temperature. RNase Inhibitor and dNTP mix can be purchased from SYBR Green qPCR Mix (2X) , SYBR Green qPCR Mix (2X, Low ROX) , and SYBR Green qPCR Mix (2X, High ROX) are for conventional qPCR analysis with SYBR Green fluorescent dye. Probe qPCR Mix (2X) , Probe qPCR Mix (2X, Low ROX) , and Probe qPCR Mix (2X, High ROX) are for qPCR analysis with Taqman Probe.b. Perform qPCR analysis following the instructions of the corresponding qPCR kit.3. For other applications such as primer extension and probe labeling, please refer to references related to M-MLV reverse transcriptase.FAQ:1. The reverse transcription product of total RNA is invisible after electrophoresis.It is a normal phenomenon, because the amount of RNA template is low, and the amount of reverse transcription products in different sizes is even lower. 2. No specific product is 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 the target gene are not well designed. 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 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, spermidine, etc, which can be removed effectively by column purification or precipitation, washing, and redissolution. Total RNA extracted by Zol or Trizol produced by can fully meet the requirements of reverse transcription.d. Insufficient templates for reverse transcription. When DNase I is used to remove the residual DNA in the RNA sample prior to reverse transcription and is subjected to heat-inactivation, EDTA should be added to the 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. An 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.
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