The-MIQE-Guidelines-MIQE指南
关于欧盟GMP指南附录11“计算机系统” 的修订 - 概念文件介绍
Revision of the EU GMP Guide Annex 11 "Computerised Systems" -Presentation of Concept Paper关于欧盟GMP指南附录11“计算机系统”的修订- 概念文件介绍The current EU GMP Guidance Annex 11 "Computerised Systems" has been in force since 2011. It has been discussed for a long time to revise this annex in order to meet current technological and regulatory developments. On 16 November 2022, the EMA (European Medicines Agency) published a 5-page "Concept-Paper on the revision of Annex 11 of the guidelines on Good Manufacturing Practice for medicinal products -Computerised Systems". Comments on this concept paper can be submitted until 16 January 2023.当前的欧盟GMP指南附录11“计算机化系统”自2011年起就已经生效。
关于修订该附录以反映最新的技术和法规发展的讨论，已经持续了很长一段时间。
2022年11月16日，EMA（欧洲药品管理局）发表了一份长达5页的“关于修订药品良好生产规范指南附录11-“计算机化系统”的概念文件”。
有关此概念文件的评论可以持续提交，截止到2023年1月16日。
罗氏RNA提取试剂盒使用指南说明书
Step 1: Sample Preparation & Nucleic Acid IsolationFor great results, use (click product names to learn more):Roche High Pure RNA Isolation KitRoche High Pure FFPET RNA Isolation KitRoche High Pure miRNA Isolation KitRoche RealTime ready Cell Lysis KitFrom which source (animal, organ, tissue) does the examined material originally come from? Which volume or mass or cell number was used for nucleic acid preparation?My MIQE Guide*Empowering results that matter Sponsored by Roche Applied Science Experiment title:Performed by:Date:Institution:Experimental design: How did you choose and set up your study (number of treated samplesand controls)Handling: Which tools or methods were used to obtain and process the primary samples (e.g., micro-dissection, macrodissection)?Method of processing and preservation: How was the sample treated and stored?If frozen – how and how quickly?If fixed – with what, and how quickly?If stored for longer: how and how long? (especially for FFPE samples)Extraction method:Which kit or instrument was used to extract/isolate the DNA/RNA from the starting material? Roche High Pure RNA Isolation Kit, High Pure FFPET RNA Isolation Kit, High Pure miRNA Isolation Kit, RealTime ready Cell Lysis Kit, or other (Please specify)Was the vendor’s protocol modified (If Yes, when, and how? e.g. by using additives)Did you do a DNAse or RNAse treatment? (If Yes, when?)Did you check for nucleic acid purity and integrity? If Yes: By using which instrument and method? What was the resulting purity (A260/A280)? What was the resulting yield? If No: Why not?Did you check for the presence of PCR inhibitors? If Yes: By using what (e.g. Cq dilutions, spike or other (please specify)If No: Why not?Final storage solution (e.g., buffer, H2O) for the purified total RNA:Storage time and temperature of the purified total RNA before use in RT-qPCR:Step 2:Reverse TranscriptionFor optimal results, use:Roche Transcriptor First Strand cDNA Synthesis KitRoche Transcriptor Universal cDNA MasterAmount of RNA and reaction volume:Priming oligonucleotide (if using gene specific primers) and concentration: Reaction temperature and time:Manufacturer of reverse transcription reagent(s) and catalogue number(s): Reverse transcriptase type and used concentration:Storage conditions of cDNA:Step 3:PCR Amplification and AnalysisFor best results, use:LightCycler® 480 Probes MasterFastStart Essential DNA Probes MasterFastStart Universal Probe Master (Rox)Target sequence and amplicon information: Target gene database sequence accession number:Location of amplicon:Amplicon length:Result of in silico specificity screen (BLAST, etc.):Information on pseudogenes, retropseudogenes or other homologs: Secondary structure analysis of amplicon:Determined by which method?Location of each primer relative to exons or introns (if applicable): Targeted splice variants:RTPrimerDB Identification Numbers: Manufacturer of oligonucleotides: Purification method:For probe-based assays: Probe type:qPCR reaction conditionsReaction volume and amount of cDNA/DNA per reaction: Primer, (probe), Mg2+ and dNTP concentrations: Polymerase identity:Buffer/kit manufacturer and identity (e.g., catalog number)Manufacturer and catalog number of plates or tubes and catalog number:Complete thermocycling parameters:Reaction setup: Was it manual or robotic? If robotic: Using which robot?Equipment: Which Real-Time PCR instrument was used? (Which Roche LightCycler® System or other (please specify)?)Validation of qPCR runs:Are you running a multiplex assay? If yes, please describe efficiency and limit of detection foreach assay:How did you check for specificity of amplification for each target (e.g., on a gel, by sequencing, melt-ing curve analysis or digest):For SYBR Green I assays: Cq of the non-template control reaction:Standard curve characteristics (slope and y-intercept):How many replicates did you use to establish the standard curve?(xx replicates per standard concentration)What was the lower and the upper limit of the standard curve?PCR efficiency calculated from slope:Confidence interval for PCR efficiency or standard error:r2 of standard curve:Information on linear dynamic range:Cq variation at lower limit: Confidence intervals throughout range:Evidence for limit of detection:How many reactions per run were used for controls? (please specify positive and negative controls, controls without template and No RT controls, e.g. Positive controls: 3 reactions in 5 replicates per 96 well plate)Data analysis:Vendor software: Which software type, version and algorithm provided by the PCR machine supplier was used to analyze the data?Specialist software: Which (if any) additional software was used? Self-developed algorithms,or other (please specify)Normalisation: Which reference gene(s) were used to calculate the relative expression of the studied genes?What was the reason for choosing these particular genes?Which algorithm (e.g., geNorm, bestkeeper, normfinder) was used to normalize for reference gene(s)Which principle was used for Cq calling?What was the number and of biological replicates used?How was their concordance?How many technical replicates were used, and at which step (RT or qPCR)? What was the observed repeatability (intra-assay variation)?What was the observed reproducibility (inter-assay variation, %CV)The MIQE guidelines empower results that truly matter. And so does Roche.Visit to discover all the materials you need for truly remarkable research results.* modified based on the list in the original MIQE guidelines publication with permission of the MIQE authors.For life science research only. Not for use in diagnostic procedures. LIGHTCYCLER and FASTSTART are trademarks of Roche.All other product names and trademarks are the property of their respective owners. NOTICE: This product may be subject to certain use restrictions. Before using this product, please refer to the Online Technical Support page () and search under the product number or the product name, whether this product is subject to a license disclaimer containing use restrictions.Published byRoche Diagnostics GmbH Sandhofer Straße 116 68305 Mannheim Germany© 2013 Roche Diagnostics. All rights reserved.*********** 1012。
RECSIT1.1中英文对照全文
New response evaluation criteria in solid tumours: Revised RECIST guideline (version 1.1)新版实体瘤疗效评价标准：修订的RECIST指南（1.1版本）Abstract摘要Background背景介绍Assessment of the change in tumour burden is an important feature of the clinical evaluation of cancer therapeutics: both tumour shrinkage (objective response) and disease progression are useful endpoints in clinical trials. Since RECIST was published in 2000, many investigators, cooperative groups, industry and government authorities have adopted these criteria in the assessment of treatment outcomes. However, a number of questions and issues have arisen which have led to the development of a revised RECIST guideline (version 1.1). Evidence for changes, summarised in separate papers in this special issue, has come from assessment of a large data warehouse (>6500 patients), simulation studies and literature reviews.临床上评价肿瘤治疗效果最重要的一点就是对肿瘤负荷变化的评估：瘤体皱缩（目标疗效）和病情恶化在临床试验中都是有意义的判断终点。
药品共线生产质量管理指南英文
药品共线生产质量管理指南英文English answer:Quality Management Guidelines for Co-line Production of Pharmaceutical Products.Introduction.Co-line production is a manufacturing process in which multiple products are produced on the same production line. This can be a cost-effective way to produce products, butit also poses some unique challenges to quality management.Quality Management System.The quality management system (QMS) for co-line production must be designed to ensure that all products are produced in accordance with their respective specifications. The QMS should include the following elements:A quality policy that defines the company's commitment to quality.A quality manual that describes the QMS.Standard operating procedures (SOPs) that describe how specific processes are to be performed.A quality assurance (QA) program to monitor and audit the QMS.A quality control (QC) program to test products and ensure that they meet specifications.Product Development.The product development process for co-line production must be carefully managed to ensure that products are compatible with each other and with the production line. The following steps should be taken during product development:Conduct a risk assessment to identify potential risks associated with co-line production.Develop a co-line production plan that outlines how products will be produced on the same line.Validate the co-line production plan to ensure that it is effective.Conduct a stability study to ensure that products are stable when produced on the same line.Manufacturing.The manufacturing process for co-line production mustbe carefully controlled to ensure that products are produced in accordance with their respective specifications. The following steps should be taken during manufacturing:Use dedicated equipment for each product.Clean and sanitize equipment between products.Follow SOPs for each process.Inspect products at critical control points.Test products to ensure that they meet specifications.Quality Assurance.The QA program for co-line production must be designedto monitor and audit the QMS to ensure that it is effective. The following steps should be taken during QA:Conduct regular audits of the QMS.Review product quality data.Investigate and resolve any quality issues.Quality Control.The QC program for co-line production must be designedto test products and ensure that they meet specifications. The following steps should be taken during QC:Test products at critical control points.Conduct stability testing.Release products for distribution only if they meet specifications.Conclusion.Co-line production can be a cost-effective way to produce pharmaceutical products, but it also poses some unique challenges to quality management. By following the guidelines outlined in this document, companies can ensure that they produce safe and effective products while minimizing the risk of cross-contamination and other质量问题.中文回答：药品共线生产质量管理指南。
MIQE指南
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Fleige S and Pfaffl MW (2006). RNA integrity and the effect on the real-time qRTPCR performance. Mol Aspects Med 27, 126-139.
Garson JA et al. (2009). Unreliable real-time PCR analysis of human endogenous retrovirus-w (HERV-W) RNA expression and DNA copy number in multiple sclerosis. AIDS Res Hum Retroviruses 25, 377-378; author reply 379-381.
Bio-Rad’s real-time thermal cyclers are licensed real-time thermal cyclers under Applera’s United States Patent 6,814,934 B1 for use in research, human in vitro diagnostics, and all other fields except veterinary diagnostics. This product is covered by one or more of the following U.S. patents or their foreign counterparts owned by Eppendorf AG: U.S. Patent Nos. 6,767,512 and 7,074,367.
MIQE指南范文
MIQE指南范文MIQE（Minimum Information for Publication of Quantitative Real-Time PCR Experiments）指南是一个旨在提高实时定量PCR（qPCR）实验的透明度和可重复性的指南。
下面是一篇关于MIQE指南的范文，字数超过1200字：Abstract:Introduction:Material and Methods:1. Sample Collection: Tissue samples from different organs were collected from ten healthy individuals. The samples were immediately frozen in liquid nitrogen to preserve RNA integrity.2. RNA Extraction: Total RNA was extracted from the tissue samples using the TRIzol reagent following the manufacturer's instructions. RNA concentration and quality were determinedusing a spectrophotometer.3. cDNA Synthesis: RNA samples were treated with DNase I to remove genomic DNA contamination. cDNA was synthesized using the SuperScript III Reverse Transcriptase kit following the manufacturer's protocol.5. qPCR Assay: qPCR amplifications were performed using the SYBR Green PCR Master Mix and the Applied Biosystems 7500 Real-Time PCR System. The cycling conditions consisted of an initialdenaturation at 95°C for 10 minut es, followed by 40 cycles of 95°C for 15 seconds and 60°C for 1 minute.6. Data Analysis: The cycle threshold (Ct) values were determined using the Applied Biosystems software. The relative expression levels of Gene X were calculated using the 2^-ΔΔCt method, with GAPDH as the reference gene.Results:1. RNA Concentration and Quality: The concentration and purity of the RNA samples were assessed using a spectrophotometer. The samples exhibited an A260/A280 ratioof >1.8, indicating high RNA purity.2. Primer Specificity and Efficiency: The primer specificity was confirmed by the presence of a single peak in the melting curve analysis and the absence of non-specific amplification products. The primer efficiency was determined using a standard curve, which exhibited a slope of -3.32, corresponding to an efficiency of 97.9%.Discussion:Conclusion:In conclusion, following the MIQE guidelines is crucial for conducting accurate and reproducible qPCR experiments. This study demonstrated the successful application of the MIQE guidelines in investigating the expression of Gene X in varioustissues and conditions. By adhering to these guidelines, we were able to obtain reliable data, contributing to a better understanding of gene expression regulation.。
MIQE指南范文
MIQE指南范文MIQE指南是科研实验中遵循的一项质量标准，全称为“Minimum Information for Publication of Quantitative Real-Time PCR Experiments”，直译为“发表定量实时聚合酶链反应实验证据的最低信息要求”。
它是为了提高定量实时聚合酶链反应（qPCR）实验的质量与可重复性而制定的指南。
MIQE指南包含了一系列涵盖所有与qPCR实验相关的信息要求，以确保实验与结果的准确性和可重复性。
它提供了一个标准化的实验报告模板，研究人员可以按照这个模板来组织和记录他们的实验过程和结果。
MIQE指南也为实验室提供了一些推荐的实验操作规范和技术流程，以确保实验过程的一致性和标准化。
2.试剂和材料：需要提供准确的实验试剂和材料的详细信息，包括生产商、批号和纯度等。
3.实验设计和数据分析：需要提供实验的设计和数据分析方法，包括实验方案、控制组设置、技术重复次数等。
4.仪器和反应条件：需要提供所使用的qPCR仪器的详细信息，包括型号和硬件配置，还需要提供反应条件等信息。
5.正确性和灵敏度：需要提供实验中用于验证方法准确性和灵敏度的相关信息，例如限制性酶切、序列分析等。
6.结果和讨论：需要提供实验结果的详细信息，以及对结果的客观分析和讨论，包括重复性和统计学分析等。
MIQE指南的目标是确保qPCR实验在不同实验室之间的结果是可比较和复制的，有助于科学研究的可靠性和可重复性。
同时，MIQE指南也使得科学家能够更好地了解实验操作的细节，以便更好地理解和解释结果，并且可以帮助实验设计和分析方法的改进。
总之，MIQE指南是为了提高定量实时聚合酶链反应实验的质量和可重复性而制定的一项质量标准指南。
它要求研究人员提供详细的实验描述和结果分析，以确保实验和结果的准确性和可比较性。
MIQE指南对于qPCR实验的标准化和科学研究的可靠性和可重复性具有重要意义。
MIQE Guidelines
/pcr
AMPLIFICATION
Nucleic Acid Extraction （核酸提 取）
/pcr
RNA 纯度和完整性
AMPLIFICATION
Experion Virtual Gel
L C 3’ 5’ 10’ 15’ 1h 2h 4h
Primer B
Forward Primer
Reverse Primer A
1
110
/pcr
Amplicon Secondary Structures
AMPLIFICATION
/mfold/applications
编辑们犯难了“定量PCR数据可信吗？”
/pcr
What are the MIQE guidelines?
AMPLIFICATION
qPCR的国际标准：就评价qPCR实验和发表文章时所必需的实验 信息提出了最低限度的标准。
/pcr
MIQE 指南好处
AMPLIFICATION
/pcr
Reverse Transcription 反转录
AMPLIFICATION
/pcr
Reverse Transcription
AMPLIFICATION
RNA
cDNA
Reality Ideal ?
Reproducible Data Not Reproducible
/pcr
qPCR Target Information
AMPLIFICATION
/pcr
序列同源性分析(BLAST)
AMPLIFICATION
http://www.ncbi.nlm.nih. gov/BLAST/Blast.cgi
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qPCR
• 二级结构：mfold • 特异性：BLAST，验证（电泳凝胶、解链曲线图形、DNA测序） • 引物优化：退火温度和镁离子梯度、Cq值、荧光点数对比循环次数和溶解曲线 • 质控品和定量校准品：每一块反应板都要有NTC并确立数据排除标准 • 分析性能：PCR的效率——校准曲线法（斜率、Y轴截距）
应该有稳定表达，且丰度与样本中mRNA总量有较强相关性。 • 生物系统固有变异：大量样本以增加统计学意义。 • 质量分析：定性PCR也需要提供其分析性能的详细内容（线性动
态范围和检测限）。
总结
项目 实验设计
样品
核酸提取
逆转录
必要
实验组和对照组的定义 各组组内编号
采样方式 处理过程（是否冰冻/固定）
概念
• 分析灵敏度：单次分析中能够准确测定的样本的最小拷贝数（浓度），区别于 临床灵敏度。
• 分析特异性：qPCR分析在样本中有其他物质存在时只检测目标序列的能力。区 别于诊断特异性。
• 准确度：实验测定浓度与实际浓度的差异，以差异倍数和拷贝数的估计值表示。 • 重复性（短期/批内）：同一方法分析同一样本的精密度和耐用性，Cq的变异。 • 重现性（长期/批间）：批间结果或不同实验室间结果的变异，拷贝数的变异。 • 描述目标基因mRNA浓度：RT-PCR分析+引物序列信息、引物相对于特定剪接变
MIQE指南：发表实时荧光定量
PCR实验所需提供的最低信息量
钟艺 2014.12.15
背景
• 缺乏共识 • 细节不够
目的
• 使作者能够设计和报告更有价值的qPCR实验。 • 使评阅人员和编辑按照相应的标准来评价所提交的文稿的技术质
量。 • 使读者更容易重复按照该指南发表的实验研究。
学术术语
• qPCR——实时定量PCR（Quantitative Real-time PCR） • RT – PCR——反转录PCR（Reverse Reaction PCR） • 标准化基因——参照基因（reference genes） • TaqMan探针——水解探针（Hydrolysis Probes） • FRET探针——荧光共振能量转移探针 • LightCycler型探针——双杂交探针 • 定量——quantification • 循环数——Cq（quantification cycle）
添加剂（SYBR Green I，DMSO等） qPCR仪仪器制造商
非必要
扩增子位置 假基因，反转录假基因，同系物
序列比对 扩增子的二级结构分析
针对什么剪接体
RTPrimerDB中的识别号 探针序列 引物制造商 纯化方法
缓冲液的化学成分 反应管/板的商品编号及制造商
PCR循环方式（手动/自动）
总结
线性动态范围——平均校准曲线法，动态范围至少跨越3个数量级，浓度范 围达到5~6个log10浓度，线性区间包括目标核酸的定量范围，报告最低浓度的变异与相 关系数（R2值），提供整个线性动态范围内的CI（可信区间）值。 • 检测限：能够检测到95%的阳性标本的最低浓度。 • 精密度：进行多次重复测定，并标示重复性（SD）和可信区间。对于诊断分析还需报
• 简单描述样本：肿瘤显微镜活检——活检标本中肿瘤细胞组成的 百分比。
• 核酸提取：提取方法、测定浓度的方法、质量评价方法
核酸的质量控制——RNA样本
• RNA样本：保证各样本中含有大致相同量的RNA 检测和报告全基因组DNA的污染程度 RNA样品是否已经过RNA酶处理（酶类型及反应条件） 核酸目标在阳性质控品和NTC下Cq值的比较 RNA模板的质量评价——数量、污染情况、完整度 A260/280——中性PH的缓冲盐 测定mRNA浓度微小（<10X）差异：凝胶电泳
项目
必要
qPCR验证
特异性（凝胶电泳、测序、溶解曲线、酶切） NTC的Cq
告不同地点和不同操作者的批间精密度（重现性）。 • 多重qPCR：需证明在多重PCR下的分析效率及LOD与单一PCR反应下相同。
数据分析
• 提供数据分析方法和置信限评估的详细信息及分析软件的性能指 标。
• 标准化：参考基因的选择优化及最佳数量 不提倡采用单一参考基因，除非能证明其在所述实验条
件下没有表达差异。 报告目的基因参考基因mRNA浓度的比值，参考基mRNA
储存条件及时间
提取方法和仪器及试剂盒名称 DNA酶和RNA酶处理的细节 DNA或RNA污染 核酸含量及测定方法和仪器
RNA完整性及测定方法和仪器 PCR抑制性检测（样本稀释法）
反应条件 RNA量和反应体系
引物及其浓度 逆转录酶及其浓度
温度和时间
非必要 研究者所在实验室
致谢
样品体积与质量
添加剂来源 核酸纯度（A260/A280）
• DNA样本：阳性质控——检测抑制作用
反转录
• 实验方案和试剂 RNA量、引物、酶的类型、酶的用量、温度、反应时间
• 2~3次重复（相同RNA浓度）
qPCR
• 每个目的基因和对照基因的数据库检索编号 • 每个引物和探针的外显子位置 • 寡核苷酸的序列和浓度（染料或被修饰的碱基的性质、位置和连接） • 聚合酶的浓度和性质 • 模板DNA或cDNA的量 • 镁离子的浓度 • 缓冲盐的化学组成（盐、PH值和添加剂）及反应体积 • qPCR仪和循环条件 • 器材制造商 • 塑料器皿透明度（白色/透明） • 密封方式（热合/粘合）
核酸产量 电泳结果
试剂制造商及目录编号 cDNA的储存
总结
项目 qPCR目的基因信
息
qPCR引物
qPCR程序
必要
基因标志 序列登陆号 扩增子长度 软件的特异性筛选（BLAST） 各引物的外显子或内含子位置
引物序列 所有结合位点
完整的反应条件及热循环参数 反应体积和模板量
引物（探针），Mg2+，dNTP浓度 聚合酶识别位点及其浓度 缓冲液或试剂盒制造商
异体的特异性评价信息、有文件证明的转录过程中单核苷酸多态性的位置信息、 使用的单核苷酸多态性数据库。（RTprimerDB） • mRNA浓度 ≠ 蛋白浓度
研究ห้องสมุดไป่ตู้诊断
• 研究样本：来源广泛、低通量、样品类型不同 • 诊断样本：数量有限、高通量、样品类型少
样本的采集、处理和制备
• 待测目标为RNA——组织样本的采集来源、是否立即处理、保存 时间、保存条件；