Table of Contents 目录 Introduction 1 Limitations of the Product 1 Materials Provided and Storage 3 Other Supplies Required 3 Precaution 5 QC Testing 5

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Catalog Number MK0601-025 MK0601-100 For cdna synthesis with integrated removal of genomic DNA

1 This package insert must be read entirely before using this product. For proper performance, use the insert provided with each individual product received. Catalog Number MK MK For cdna synthesis with integrated removal of genomic DNA contamination. 适用于合成 cdna, 并去除基因组 DNA 污染 cdna Reverse Transcription Kit cdna 反转录试剂盒 For research use only. Not for use in diagnostic procedures. 仅用于科研, 不得用于临床诊断 05/2017

2 Table of Contents 目录 Introduction 1 Limitations of the Product 1 Materials Provided and Storage 3 Other Supplies Required 3 Precaution 5 QC Testing 5 Protocol 7, 9 Troubleshooting 11, 13, 15, 17 Ordering Information 19 产品介绍 2 产品的局限 2 提供的材料及贮存 4 未提供的材料设备 4 注意事项 6 质控 6 实验流程 8, 10 常见问题与解决方案 12, 14, 16, 18 订购信息 20

3 INTRODUCTION The cdna Reverse Transcription Kit provides a fast and convenient procedure for effective genomic DNA elimination and efficient reverse transcription, which takes as little as 30 minutes. The kit is dedicated for use in reverse-transcription PCR and real-time PCR. Reverse Transcriptase has a high affinity for RNA and is optimized for efficient and sensitive cdna synthesis from 50 ng to 2 μg of RNA. This high RNA affinity enables high cdna yields, even from templates with high GC-content or complex secondary structure. RT Primer Mix ensures cdna synthesis from all regions of RNA transcripts, even from 5' regions. This allows high yields of cdna template for real-time PCR analysis regardless of where the target region is located on the transcript. The cdna Reverse Transcription Kit comprises 2 main steps: elimination of genomic DNA and reverse transcription. The purified RNA sample is briefly incubated at 42 for 2 minutes to effectively remove contaminating genomic DNA. In contrast to other methods, the RNA sample is then used directly in reverse transcription. The reverse transcription reaction takes place at 42 and is then inactivated at 95. In contrast to other methods, additional steps for RNA denaturation, primer annealing, and RNase H digestion are not necessary. The synthesized cdna is ready for use in downstream applications, or can be stored at -20 for later use. 产品介绍 cdna 反转录试剂盒提供快速简单的方法有效地去除基因组 DNA 并高效地进行逆转录, 整个过程仅需 30 分钟本试剂盒可用于逆转录 PCR 和实时 PCR 逆转录酶与 RNA 具有高亲和力, 可将 50 ng - 2 μg 的 RNA 有效地进行 cdna 合成对 RNA 的高亲和力可保证 cdna 的高产量, 即使模板中的 GC 含量高或具有复杂的二级结构 RT Primer Mix 可使 cdna 从 RNA 转录本的各个区域 ( 即使是 5 区 ) 进行合成因此, 无论目标区域位于转录本的哪一段, 本试剂盒均可获得高产的 cdna 模板用于实时 PCR 检测 cdna 反转录试剂盒包含 2 个主要步骤 : 去除基因组 DNA 和逆转录纯化的 RNA 样本在 42 孵育 2 分钟, 有效去除基因组 DNA 污染与其它方法不同的是, 处理后的 RNA 可直接用于逆转录逆转录反应在 42 进行, 随后在 95 灭活与其它方法不同, 此次无需额外的 RNA 变性引物退火和 RNA 酶 H 消化合成的 cdna 可即刻用于下游实验或保存于 -20 待后续使用 LIMITATIONS OF THE PRODUCT The product is intended for molecular biology applications. FOR RESEARCH USE ONLY. NOT FOR USE IN DIAGNOSTIC PROCEDURES. The product should not be used beyond the expiration date on the label. Do not mix reagents with those from other lots or sources. 产品的局限 1. 本产品用于分子生物学实验, 仅用于科学研究, 非诊断试剂, 不能用于临床诊断 2. 请在本产品标记的有效期内使用 3. 本产品的试剂不能与其他批号的试剂或其他来源的试剂混合使用 1 2

4 MATERIALS PROVIDED AND STORAGE 提供的材料和贮存 Components Product Code gdna Buffer MR μl 220 μl RNase-Free water MR ml 1.5 ml 2 5 RT Buffer MR μl 420 μl RT Enzyme Mix MR μl 220 μl RT Primer Mix MR μl 420 μl 组分编号 gdna 缓冲液 MR μl 220 μl 无 RNA 酶水 MR ml 1.5 ml 2 5 逆转录缓冲液 MR μl 420 μl 逆转录酶混合物 MR μl 220 μl 逆转录引物混合物 MR μl 420 μl Note: All reagents can be stored at - 20 for up to 1 year. 注 : 所有试剂可在 - 20 最多保存 1 年 OTHER SUPPLIES REQUIRED 1. Ice 2. RNase-free PCR tubes 3. Pipet tips (Sterile, RNase-free) 4. PCR thermal cycler 5. Microcentrifuge (with rotor for PCR tubes) 6. Thermostat water bath or Thermomixer 7. Vortexer 未提供的材料设备 1. 冰 2. 无 RNA 酶的 PCR 管 3. 吸头 ( 无菌, 无 RNA 酶 ) 4. PCR 热循环仪 5. 微量离心机 ( 配 PCR 管的转子 ) 6. 恒温水浴锅或恒温混匀仪 7. 振荡器 3 4

5 PRECAUTION 1. Reagents are intended for research use only and are not for use in diagnostic or therapeutic procedures. 2. Do not mix or substitute reagents with those from other lots or other sources. 3. Do not use reagents beyond expiration date on label. 4. We therefore recommend that this product is handled only by those persons who have been trained in laboratory techniques and that it is used in accordance with the principles of good laboratory practice. Wear suitable protective clothing such as laboratory overalls, safety glasses, mask and gloves. 5. Always wear latex or vinyl gloves while handling reagents and RNA samples to prevent RNase contamination from the surface of the skin or from dusty laboratory equipment. Change gloves frequently and keep tubes closed whenever possible. 6. Do not eat or smoke in areas where reagents or samples are handled. 7. The use of sterile, disposable polypropylene tubes is recommended throughout the procedure. These tubes are generally RNase-free and do not require pretreatment to inactivate RNases. 8. It is important to have intact RNA as starting template. Even trace amounts of contaminating RNases in the RNA sample can cause RNA cleavage, resulting in shortened cdna products. 9. Set up all reactions on ice to minimize the risk of RNA degradation. 10. For RNA with a very high degree of secondary structure, we recommend a 5-minute s incubation at 65,then transfer to ice quickly before reaction. 11. RT Primer Mix contains both oligo-dt and random primers, if only oligo-dt or gene-specific primer is needed instead of RT Primer Mix, add 50 pmol oligo-dt or 5 pmol gene-specific primer in a 20 μl reaction, adjust the volume of primer to 4 μl with RNase-free water. 12. Chemical impurities, such as protein, poly-anions (e.g., heparin), salts, EDTA, ethanol, and phenol, can affect the activity and processivity of the reverse transcriptase. 注意事项 1. 本产品用于科学研究, 不能用于诊断治疗 2. 请不要使用其他批号或其他来源的试剂替代本产品中的试剂 3. 请不要使用过期的试剂 4. 联科生物推荐只有经过良好实验室培训的工作人员方可操作本产品操作时请穿戴合适的防护设施, 例如实验服安全眼镜口罩和手套等 5. 操作试剂和 RNA 样本时, 始终穿戴乳胶或乙烯手套, 防止来自皮肤表面或肮脏的实验设备的 RNA 酶污染如有可能, 经常更换手套并保持管口紧闭 6. 在操作试剂或处理样本的区域请不要饮食 7. 整个过程中推荐使用无菌一次性聚乙烯管, 通常这些离心管是无 RNA 酶的, 无需预处理 8. 完整的 RNA 作为起始模板非常重要微量的 RNA 酶会剪切 RNA, 产生变短的 cdna 产物 9. 在冰上进行操作, 以降低 RNA 降解的风险 10. 对于二级结构非常复杂的 RNA, 我们推荐在反应前先将模板在 65 孵育 5 分钟, 随后迅速转移至冰上 11. RT Primer Mix 中包含了 oligo-dt 和随机引物, 如果需要单独使用 oligo-dt 或基因特异性引物, 在 20 μl 反应体系中加入 50 pmol oligo-dt 或 50 pmol 基因特异性引物, 并用无 RNA 酶水调整体积至 4 μl 12. 化学杂质如蛋白聚阴离子如肝素盐离子 EDTA 乙醇和酚, 会影响逆转录酶的活性和持续性 QC Testing Each lot of MultiSciences product is carefully tested in the quality control team. 质控联科生物每个批次的产品都由质控部门进行严格的检测 5 6

6 PROTOCOL Important points to know The protocol is optimized for use with 50 ng to 2 μg of RNA. If using > 2 μg RNA, scale up the reaction linearly to the appropriate volume. 实验流程实验须知本实验方案针对模板量为 50 ng - 2 μg 的 RNA 而设计, 如模板量大于 2 μg, 请按比例扩大反应体积 Procedure 1. Thaw template RNA on ice. Thaw 5 gdna Buffer, 5 RT Buffer, RT Primer Mix and RNase-free water at room temperature. Mix each solution by vortexing. Centrifuge briefly to collect residual liquid from the sides of the tubes, and then store on ice immediately. 2. Prepare the genomic DNA elimination reaction on ice according to Table 1. Mix by short centrifugation, incubate for 3 minutes at 42. Then place immediately on ice. Table 1. Genomic DNA elimination reaction components Component Volume/reaction 5 gdna Buffer 2 μl Template RNA x μl RNase-Free Water (8 - x) μl Total volume 10 μl 3. Prepare the reverse-transcription mixture on ice according to Table 2. Note: If setting up more than one reaction, prepare a volume of mixture 10 % greater than that required for the total number of reactions to be performed. Table 2. Reverse-transcription reaction components Component Volume/reaction 5 RT Buffer 4 μl RT Enzyme Mix 2 μl RT Primer Mix 4 μl Total volume 10 μl 操作步骤 1. 将模板 RNA 在冰上融化将 5 gdna Buffer 5 RT Buffer RT Primer Mix 和 RNase-free water 在室温融化涡旋混匀每种溶液, 简短离心收集残留在管壁的液体, 立即置于冰上 2. 根据表 1 在冰上制备基因组 DNA 去除反应体系简短离心,42 孵育 3 分钟, 随后立即置于冰上表 1. 基因组 DNA 去除反应体系组分组分每个反应的体积 5 gdna Buffer 2 μl Template RNA x μl RNase-Free Water (8 - x) μl 总体积 10 μl 3. 根据表 2 在冰上制备逆转录混合物注意 : 如果要进行多个反应, 根据反应总数计算所需的理论体积, 并额外多制备 10 % 的体积表 2. 逆转录反应体系组分组分每个反应的体积 5 RT Buffer 4 μl RT Enzyme Mix 2 μl RT Primer Mix 4 μl 总体积 10 μl 7 8

7 4. Distribute 10 μl of mixture prepared in step 3 into individual reaction tubes prepared in step 2. Mix well and centrifuge briefly to collect residual liquid from the sides of the tubes. 5. Incubate at 42 for 15 minutes. Note: This step could be completed on PCR thermal cycler. 6. Incubate at 95 for 3 minutes to inactivate Reverse Transcriptase. Store reversetranscription reactions on ice and proceed directly with downstream applications, or for long-term storage, store at -20. Note: This step could be completed on PCR thermal cycler. 4. 取步骤 3 中配制的 10 μl 混合物加入至步骤 2 所制备的反应管中混匀, 简短离心收集残留在管壁的液体孵育 15 分钟注意 : 此步可在 PCR 热循环仪上完成孵育 3 分钟以灭活逆转录酶将逆转录反应物置于冰上, 直接进行下游实验, 或 -20 长期保存注意 : 此步可在 PCR 热循环仪上完成 9 10

8 Troubleshooting 常见问题与解决方案 No product, or product detected late in real-time PCR (problems occurring during reverse transcription) a) Pipetting error or missing Check the pipets used for experimental setup. Mix all reagents reagent when setting up well after thawing, and repeat the reverse-transcription reaction. reverse-transcription reaction b) Incorrect setup of reverse- Be sure to set up the reaction on ice. transcription reaction Adding too much reverse-transcription reaction to the PCR mix c) Too much reverse-transcription may reduce amplification efficiency and the linearity of the reaction added reaction. Generally, the volume of reverse-transcription reaction added should not exceed 10% of the final PCR volume. Reverse transcription should be carried out at 42. Check the d) Temperature of reverse- temperature of heating block or water bath. Generally, transcription reaction temperatures > 42 will reduce the activity of reverse transcriptase and therefore affect the cdna yield. The standard reverse-transcription reaction requires a 15-minute incubation. In rare cases, when analyzing RNAs with a very e) Short incubation time high degree of secondary structure or if the RT-PCR product is longer than 200 bp, it may be advantageous to increase the incubation time to 30 minutes. Check the concentration, integrity, and purity of the template f) Poor quality or incorrect RNA before starting the protocol. Mix well after thawing the amount of template RNA for template RNA. Even trace amounts of RNases can affect reverse-transcription reaction synthesis of cdna and sensitivity in RT-PCR, particularly with small amounts of RNA. This kit is designed for use with 50 ng to 2 μg RNA. If using > g) RNA concentration too high 2 μg RNA, scale up the reaction linearly to the appropriate or too low volume. Denaturation of the template RNA is not necessary. If h) RNA denatured denaturation was performed, the integrity of the RNA may be affected. 实时 PCR 检测无产物或荧光信号出现推迟 ( 逆转录过程出现的问题 ) a) 逆转录操作中, 移液出错核查使用的移液器试剂融化后混匀, 重复逆转录反应或漏加试剂 b) 逆转录反应操作不当确保在冰上开始操作 PCR 体系中加入过多的逆转录产物会降低扩增效率和反应 c) 逆转录产物加入太多线性通常加入的逆转录产物体积不应超过总体积的 10 % 逆转录应在 42 进行核查加热模块或水浴温度通常超过 d) 逆转录反应温度 42 会降低逆转录酶的活性, 从而影响 cdna 产量标准的逆转录反应需要孵育 15 分钟对于非常复杂的二级 e) 孵育时间太短结构的 RNA 或 RT-PCR 产物长度大于 200 bp, 孵育时间延长至 30 分钟可能会更好实验开始前, 核查模板 RNA 的浓度完整性和纯度融化 f) 模板 RNA 质量太差或数量后混匀即使是微量的 RNA 酶也会影响 cdna 合成以及太少 RT-PCR 的灵敏度, 尤其是在 RNA 数量较少的情况下本产品用于 50 ng - 2 μg RNA 的逆转录如 RNA 量 > 2 μg, g) RNA 浓度过高或过低请按比例放大反应体积将模板 RNA 进行变性是没有必要的如果已变性,RNA 的 h) RNA 变性完整性可能会受影响 11 12

9 i) Incorrect concentration or degradation of primers for reverse-transcription reaction j) Incubation temperature too high If using a gene-specific primer for reverse transcription, check the concentration and integrity of the primer. If necessary, perform reverse transcription with different primer concentrations or use the supplied RT Primer Mix. If using RT Primer Mix, be sure to use 1 μl of RT Primer Mix in a 20 μl reaction. Reverse transcription should be carried out at 42. Higher temperatures may reduce the length of cdna products or the activity of reverse transcriptase. Check the temperature of heating block or water bath. No product, or product detected late in real-time PCR, or only primer-dimers detected (problems occurring during real-time PCR) a) PCR annealing time too Use the annealing time specified in the protocol for the real-time short PCR kit you are using. b) PCR extension time too Use the extension time specified in the protocol for the real-time short PCR kit you are using. Always start with the Mg 2+ concentration recommended in the c) Mg 2+ concentration in PCR protocol for the real-time PCR kit you are using. Perform titration not optimal in 0.5 mm steps. d) Pipetting error or missing Check the concentrations and storage conditions of reagents, reagent when setting up including primers and cdna. PCR i) 逆转录的引物浓度不正确或已降解 j) 孵育温度过高如果使用基因特异性引物, 请核查引物的浓度和完整性如有必要, 使用不同浓度的引物或提供的 RT Primer Mix 进行逆转录如使用 RT Primer Mix, 确保每个 20 μl 反应中加入 1 μl RT Primer Mix 逆转录应在 42 进行温度过高会减少 cdna 产物长度或逆转录酶活性核查加热模块或水浴温度实时 PCR 检测无产物或荧光信号出现推迟或检测到引物二聚体 (real-time PCR 过程出现的问题 ) a) PCR 退火时间太短使用试剂盒所推荐的退火时间 b) PCR 延伸时间太短使用试剂盒所推荐的延伸时间 c) PCR 中的 Mg 2+ 浓度未优化使用试剂盒所推荐的 Mg 2+ 浓度, 以 0.5 mm 的梯度进行优化 d) PCR 操作中, 移液出错或核查试剂包括引物和 cdna 的浓度和保存条件漏加试剂 e) Taq DNA Polymerase not activated with a hot start Ensure that the cycling program includes the hot start activation step for Taq DNA polymerase; for details, check the instructions supplied with the polymerase. e) Taq 酶未热启动激活确保循环程序包括热启动激活步骤正确, 从 Taq 酶说明书中了解详情 f) PCR product too long For optimal results, PCR products should be bp in length and should not exceed 300 bp. f) PCR 产物过长 PCR 产物长度应在 bp 之间, 不超过 300 bp g) Primer design for real-time PCR not optimal Check for the presence of PCR products by gel electrophoresis or melting curve analysis. If no specific PCR products are detected, review the primer design. g) Real-time PCR 的引物未优化通过凝胶电泳或熔解曲线核查 PCR 产物的存在情况如检测不到特异性 PCR 产物, 需考虑引物的设计 h) Primer concentration for real- time PCR not optimal Use the primer concentrations recommended in the protocol for the real-time PCR kit you are using. h) Real-time PCR 的引物浓度未优化使用试剂盒所推荐的引物浓度 13 14

10 i) Insufficient number of cycles Increase the number of cycles. j) PCR annealing temperature Decrease annealing temperature in 3 steps. too high k) PCR annealing temperature too low Increase annealing temperature in 2 steps. l) No detection activated Check that fluorescence detection was activated in the cycling program. m) Wrong detection step Ensure that fluorescence detection takes place during the extension step of the PCR cycling program. n) Real-time PCR primers/ Check for possible degradation of primers/probes on a probes degraded denaturing polyacrylamide gel. o) Wrong dye layer/filter Ensure that the appropriate layer/filter is activated. chosen p) Insufficient starting template Increase the amount of template cdna, if possible. q) Primer-dimers coamplified in Include an additional data acquisition step in the cycling real-time PCR with SYBR program to avoid the detection of primer-dimers. Green I r) Detection temperature too Ensure that the detection temperature is at least 3 lower than high in optional data the Tm of the specific product. When establishing a new acquisition step for real-time primer-template system, always perform a 3-step cycling PCR with SYBR Green I reaction first, without the optional fourth step. Multiple peaks in melting temperature analysis/multiple PCR products To avoid nonspecific primer annealing, set up the real-time PCR a) Reaction set up at room in cooled reaction vessels and/or use a Taq DNA polymerase temperature which requires a hot start. High fluorescence in No RT control reactions i) 循环数不够增加循环数 j) PCR 退火温度太高以 3 梯度降低退火温度 k) PCR 退火温度太低以 2 梯度提高退火温度 l) 检测条件未激活核查循环程序中荧光检测被激活 m) 检测步骤错误确保在 PCR 循环程序中的延伸阶段进行荧光检测 n) Real-time PCR 引物 / 探针降解通过变性聚丙烯酰胺检测可能的引物 / 探针降解 o) 染料 / 通道选择错误确保选择合适的染料 / 通道 p) 起始模板量低如果可能, 增加起始模板量 q) Real-time PCR 中, 引物二聚体信号同步放大循环程序中额外增加数据获取步骤, 避免检测引物二聚体 r) Real-time PCR 中, 在可选的数据获取步骤中检测温度过高确保检测温度比特异性产物的 Tm 值低至少 3 当建立新的引物 - 模板体系时, 先进行 3 步法, 不要选择可选的第 4 步熔解曲线出现多个峰 / 多个 PCR 产物为避免非特异性的引物退火, 在低温的反应容器中进行 a) 反应在室温下操作 real-time PCR 操作, 和 / 或使用要求热启动的 Taq DNA 聚合酶在无逆转录的对照反应中出现强荧光信号 Check that the genomic DNA elimination step with gdna a) Contamination with genomic DNA Buffer was performed correctly: check the temperature of your heating block or water bath and the concentration of the reaction components. a) 基因组 DNA 污染核查使用 gdna Buffer 去除基因组 DNA 的步骤 : 加热模块或水浴温度以及反应体系中各组分浓度 15 16

11 No linearity in ratio of C T value/crossing point to log of the template amount Do not exceed maximum recommended amounts of template a) Template amount too high cdna. For details, see the protocol for the real-time PCR kit you are using. b) Template amount too low Increase amount of template RNA, if possible. High fluorescence in No Template control a) Contamination of reagents Discard reaction components and repeat with new reagents. C T 值 / 模板量的 log 交叉点不呈线性请勿超出 cdna 模板的最大推荐量从试剂盒说明书了解详 a) 模板量太高情 b) 模板量太低如有可能, 增加 RNA 模板的量无模板对照出现强荧光信号 a) 试剂污染弃去反应组分, 用新的试剂重复实验 b) Contamination during reaction setup Varying fluorescence intensity a) Real-time cycler contaminated Take appropriate safety precautions (e.g., use filter tips). Decontaminate the real-time cycler according to the supplier s instructions. b) 反应操作中出现污染使用适当的安全防护措施 ( 如使用带滤芯的吸头 ) 荧光强度变异 a) Real-time 仪器污染按照厂商说明书去除 Real-time 仪器污染 b) Real-time cycler no longer calibrated Recalibrate the real-time cycler according to the supplier s instructions. b) Real-time cycler 未校准按照厂商说明书重新校准 Real-time 仪器 17 18

12 Ordering Information Cat No. Product Name Size MK Trizol Total RNA Purification Kit I 50 T / 250 T MK Trizol Total RNA Purification Kit II 50 T / 250 T MK0201 Blood Total RNA Purification Kit 50 T / 250 T MK Animal Tissue Total RNA Purification Kit 50 T / 250 T MK0203 Cultured Cells Total RNA Purification Kit 50 T / 250 T MK0204 Plant Total RNA Purification Kit 50 T / 250 T MK Bacteria Total RNA Purification Kit 50 T / 250 T MK Viral RNA Purification Kit 50 T / 250 T MK mirna Purification Kit 50 T / 250 T MR Taq plus PCR Master Mix 1 ml MR SYBR Green PCR Mix 1 ml MR GC-Rich PCR Mix 1 ml / 5 ml MR0501 RNase-Free Water 100 ml / 500 ml NA0011 DNA Loading Buffer (6 ) 1 ml For more information about molecular reagents, please visit

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2004级研究生现代免疫学实验技术结业报告 PCR PCR PCR RT-PCR PCR (Polymerase Chain Reaction,PCR) 80 DNA, DNA PCR PCR PCR DNA PCR -- -- DNA DNA 93 DNA PCR DNA DNA ( ) DNA 55 DNA DNA -- TaqDNA dntp DNA -- -- PCR PCR DNA DNA Y (1 X) n Y DNA X (Y)