Quantitation of mRNA by Polymerase Chain Reaction: Nonradioactive PCR Methods
Quantitation of mRNA by Polymerase Chain Reaction: Nonradioactive PCR Methods
复制标题
通过聚合酶链反应定量 mRNA：非放射性 PCR 方法
DOI：
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发表时间：
1995
期刊：
影响因子：
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通讯作者：
H. Remke
中科院分区：
文献类型：
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作者：
T. Kohler;D. Lassner;A. Rost;B. Thamm;B. Pustowoit;H. Remke
使用PCR定量核酸的理论和方法前提。- 1.1 PCR核酸定量的一般情况和机会。- 1.1.1 PCR扩增模板理论。—1.1.1.1 PCR反应的数学描述。- 1.1.1.2反应的平台期。- 1.1.2 PCR定量mRNA的实验方法。—1.1.2.1外部标准品定量PCR。-滴定分析。-动力学分析。—1.1.2.2使用内标进行定量PCR。-内源性mRNA作为内部控制。-使用外源添加RNA/DNA作为内控的竞争性PCR。- 1.1.3定量RT-PCR检测的灵敏度和重复性。- 1.1.4 PCR产物的检测和定量方法。- 1.1.5避免PCR污染。- 1.2设计适合定量PCR的引物和竞争片段。- 1.2.1引物选择。- 1.2.2合成内PCR标准品的设计与构建。- 1.2.2.1作为多功能标准的合成基因（Multistandards）。- 1.2.2.2通过定点诱变构建竞争对手。- PCR方法及后续克隆策略。- PCR产品作为内部控制。- 1.2.3选择的内部标准应该是什么？——引用。- 1.3短DNA片段的克隆及体外转录生成RNA标准品。- 1.3.1理论背景。- 1.3.2实验步骤。—1.3.2.1 T/A克隆流程。- T/A克隆结扎。- T/A克隆转换。- 1.3.2.2质粒DNA的微小制备。- 1.3.2.3用限制性内切酶酶切分离质粒DNA。- 1.3.2.4 T7 RNA聚合酶体外转录克隆片段。——引用。- 1.4 PCR产物或标准品的直接非同位素测序。—1.4.1理论方面。- 1.4.2实验程序。- 1.4.2.1双链PCR产物直接非同位素循环测序。- 1.4.2.2单链PCR产物直接非同位素固相测序。——引用。- 2常规mRNA分析技术。- 2.1 mRNA的分离。- 2.1.1理论背景。- 2.1.2 RNA分离注意事项。- 2.1.3 mRNA分离方法。- 2.1.4实验步骤。- 2.1.4.1 RNAzol b法分离总rna - 2.1.4.2 Dynabeads Oligo (dT)25法纯化mRNA- 2.1.5纯化mRNA的定量。- 2.1.6纯化RNA的储存。——引用。- 2.2 cDNA的合成。- 2.2.1理论背景。- 2.2.2实验程序。- 2.2.2.1 AMV-RT逆转录酶反应。- 2.2.2.2 rth DNA聚合酶RT反应。——引用。- 2.3定性RT-PCR：合成mdr-1 cDNA的扩增。- 2.3.1理论背景。- 2.3.2实验程序。- 2.3.2.1聚合酶链反应（PCR），基本方案。- 2.3.2.2琼脂糖凝胶电泳PCR产物分析。- 2.3.2.3用限制性内切酶酶切PCR产物。- 2.3.2.4限制片段聚丙烯酰胺凝胶电泳和银染色。——引用。- 2.4单管RT-PCR。- 2.4.1理论背景。- 2.4.2实验程序。- 2.4.2.1 Taq DNA聚合酶扩增单链cDNA。- 2.4.2.2用rth DNA聚合酶扩增mdrl cDNA。——引用。- 2.5使用digi标记的DNA探针（Dot Blot）对PCR产物进行无放射性测定。- 2.5.1理论背景。- 2.5.2实验程序。- 2.5.2.1斑点印迹的制备。- 2.5.2.2印迹与dig标记探针杂交。- 2.5.2.3 DNA-DNA杂交体检测。——引用。- 2.6用dig标记的DNA探针进行非放射性Northern Blot杂交。- 2.6.1无放射性Northern Blot杂交原理及应用。- 2.6.2利用pcr生成的DNA片段制备digg标记的DNA探针。- 2.6.2.1 PCR法合成DNA片段。- 2.6.2.2 DNA片段的纯化。- 2.6.2.3随机引物对DNA片段进行dig标记。- 2.6.2.4估计dig标记DNA的产率。- 2.6.3 Northern Blots的制备。- 2.6.3.1通过变性含甲醛琼脂糖凝胶进行RNA电泳。- 2.6.3.2变性RNA到尼龙膜的毛细管转移。- 2.6.4用dig标记的DNA探针进行Northern Blot杂交。- 2.6.5 DNA-RNA杂交体的免疫学检测。-比色检测。-化学发光检测。- 2.6.6 Northern Blots分析。- 2.6.6.1 mRNA大小评估。- 2.6.6.2特异mRNA稳态水平的半定量评价。- 2.6.7非放射性检测后Northern印迹的剥离和重新探测。——引用。- 3 PCR检测核酸的半定量和定量方案。- 3.1采用ELOSA技术使用外部标准定量mRNA。- 3.1.1 ELOSA技术原理。- 3.1.2体外RNA标准PCR-ELOSA定量mrna。- 3.1.3实验步骤。- 3.1.3.1 PCR-ELOSA法定量mdr-1 mRNA。- 3.1.3.2检测的灵敏度和可重复性。——引用。- 3.2利用DNA酶免疫分析法（DEIA）进行病毒DNA的半定量检测，例如巨细胞病毒。- 3.2.1人类巨细胞病毒感染。- 3.2.2定量PCR在巨细胞病毒发病机制中的应用。- 3.2.3 CMV感染的定义。- 3.2.4巨细胞病毒感染的诊断。- 3.2.5实验程序。- 3.2.5.1 PCR样品的制备-主要困难。- 3.2.5.2人巨细胞病毒PCR检测。- 3.2.5.3 DEIA: DNA酶免疫测定。- 3.2.6 PCR诊断巨细胞病毒感染的局限性。——引用。- 3.3 hplc -核酸分析。- 3.3.1理论背景。- 3.3.2实验程序。- 3.3.2.1 PCR产物HPLC分析的好处。- 3.3.2.2 HPLC分析的缺点。——引用。- 3.4竞争性PCR法测定cDNA样本中mRNA拷贝的绝对数量。- 3.4.1理论背景。- 3.4.2实验程序。- 3.4.2.1竞争性PCR的内部DNA标准的生成。- 3.4.2.2标准寡核苷酸的纯化和校准。- 3.4.2.3竞争性PCR法定量MRP mRNA。- 3.4.2.4检测的灵敏度和可重复性。——引用。——确认。
I Theoretical and Methodical Prerequisites for Using PCR to Quantitate Nucleic Acids.- 1.1 General Aspects and Chances of Nucleic Acid Quantitation by PCR.- 1.1.1 Theory of Template Amplification by PCR.- 1.1.1.1 Mathematical Description of the PCR Reaction.- 1.1.1.2 The Plateau Phase of Reaction.- 1.1.2 Experimental Approaches to Using PCR for Quantitation of mRNA.- 1.1.2.1 Quantitative PCR Using External Standards.- Titration Analysis.- Kinetic Analysis.- 1.1.2.2 Quantitative PCR Using Internal Standards.- Endogenous mRNA as Internal Control.- Competitive PCR Using Exogenous Added RNA/DNA as Internal Control.- 1.1.3 Sensitivity and Reproducibility of Quantitative RT-PCR Assays.- 1.1.4 Methods for Detection and Quantitation of PCR Products.- 1.1.5 Avoidance of PCR Contamination.- 1.2 Design of Suitable Primers and Competitor Fragments for Quantitative PCR.- 1.2.1 Primer Selection.- 1.2.2 Design and Construction of Synthetic Internal PCR Standards.- 1.2.2.1 Synthetic Genes Serving as Multifunctional Standards (Multistandards).- 1.2.2.2 Construction of Competitors by Site-Directed Mutagenesis.- Methods Using PCR and Subsequent Cloning Strategies.- PCR Products as Internal Controls.- 1.2.3 What Should be the Internal Standard of Choice?.- References.- 1.3 Cloning of Short DNA Fragments and In Vitro Transcription to Generate RNA Standards.- 1.3.1 Theoretical Background.- 1.3.2 Experimental Procedures.- 1.3.2.1 T/A Cloning Procedure.- T/A Cloning Ligation.- T/A Cloning Transformation.- 1.3.2.2 Minipreparation of Plasmid DNA.- 1.3.2.3 Digestion of Isolated Plasmid DNA with Restriction Endonucleases.- 1.3.2.4 In Vitro Transcription of Cloned Fragments by T7 RNA Polymerase.- References.- 1.4 Direct Non-lsotopic Sequencing of PCR Products or Standards.- 1.4.1 Theoretical Aspects.- 1.4.2 Experimental Procedures.- 1.4.2.1 Direct Non-lsotopic Cyclic Sequencing of Double Stranded PCR Products.- 1.4.2.2 Direct Non-lsotopic Solid-Phase Sequencing of Single Stranded PCR Products.- References.- 2Conventional Techniques for mRNA Analysis.- 2.1 Isolation of mRNA.- 2.1.1 Theoretical Background.- 2.1.2 Precautions in RNA Isolation.- 2.1.3 Methods of mRNA Isolation.- 2.1.4 Experimental Procedures.- 2.1.4.1 Isolation of Total-RNA by RNAzol B.- 2.1.4.2 mRNA Purification by Dynabeads Oligo (dT)25.- 2.1.5 Quantitation of Purified mRNA.- 2.1.6 Storage of Purified RNA.- References.- 2.2 Synthesis of cDNA.- 2.2.1 Theoretical Background.- 2.2.2 Experimental Procedures.- 2.2.2.1 Reverse Transcriptase Reaction with AMV-RT.- 2.2.2.2 RT Reaction Using rTth DNA Polymerase.- References.- 2.3 Qualitative RT-PCR: Amplification of Synthesized mdr-1 cDNA.- 2.3.1 Theoretical Background.- 2.3.2 Experimental Procedures.- 2.3.2.1 Polymerase Chain Reaction (PCR), Basic Protocol.- 2.3.2.2 Analysis of the PCR Products by Agarose Gel Electrophoresis.- 2.3.2.3 Digesting PCR Products with Restriction Enzymes.- 2.3.2.4 Polyacryl Amide Gel Electrophoresis and Silver Staining of Restriction Fragments.- References.- 2.4 Single-Tube RT-PCR.- 2.4.1 Theoretical Background.- 2.4.2 Experimental Procedures.- 2.4.2.1 Amplification of Single-Stranded cDNA with Taq DNA Polymerase.- 2.4.2.2 Amplification of mdrl cDNA with rTth DNA Polymerase.- References.- 2.5 Nonradioactive Determination of PCR Products by Using a DIG-Labeled DNA Probe (Dot Blot).- 2.5.1 Theoretical Background.- 2.5.2 Experimental Procedures.- 2.5.2.1 Preparation of Dot Blots.- 2.5.2.2 Hybridization of the Blots with a DIG-Labeled Probe.- 2.5.2.3 Detection of DNA-DNA Hybrids.- References.- 2.6 Nonradioactive Northern Blot Hybridization with DIG-Labeled DNA Probes.- 2.6.1 Principle and Application of Nonradioactive Northern Blot Hybridization.- 2.6.2 Preparation of DIG-Labeled DNA Probes by Using PCR-Generated DNA Fragments.- 2.6.2.1 Synthesis of DNA Fragments by PCR.- 2.6.2.2 Purification of DNA Fragments.- 2.6.2.3 DIG-Labeling of DNA Fragments by Random Priming.- 2.6.2.4 Estimating the Yield of DIG-Labeled DNA.- 2.6.3 Preparation of Northern Blots.- 2.6.3.1 RNA Electrophoresis Through Denaturing Agarose Gels Containing Formaldehyde.- 2.6.3.2 Capillary Transfer of Denatured RNA to a Nylon Membrane.- 2.6.4 Northern Blot Hybridization with DIG-Labeled DNA Probes.- 2.6.5 Immunological Detection of the DNA-RNA Hybrids.- Colorimetric Detection.- Chemiluminscent Detection.- 2.6.6 Analysis of Northern Blots.- 2.6.6.1 Evaluation of mRNA Size.- 2.6.6.2 Semiquantitative Evaluation of Steady-State Levels of Specific mRNA.- 2.6.7 Stripping and Reprobing of Northern Blots After Nonradioactive Detection.- References.- 3 Semiquantitative and Quantitative Protocols for Measurement of Nucleic Acids by PCR.- 3.1 Quantitation of mRNA by the ELOSA Technique Using External Standards.- 3.1.1 Principle of the ELOSA Technique.- 3.1.2 Quantitation of mRNAs by PCR-ELOSA with External RNA Standards.- 3.1.3 Experimental Procedures.- 3.1.3.1 Quantitation of mdr-1 mRNA by PCR-ELOSA.- 3.1.3.2 Sensitivity and Reproducibility of the Assay.- References.- 3.2 Semiquantitative Detection of Viral DNA, e.g. for CMV, by Using the DNA Enzyme Immunoassay (DEIA).- 3.2.1 CMV Infection in Human Beings.- 3.2.2 Applications of Quantitative PCR in Cytomegalovirus Pathogenesis.- 3.2.3 Definition of CMV Infection.- 3.2.4 Diagnosis of Cytomegalovirus Infection.- 3.2.5 Experimental Procedures.- 3.2.5.1 Preparation of Samples for PCR - Principal Difficulties.- 3.2.5.2 Detection of Human Cytomegalovirus Using PCR.- 3.2.5.3 DEIA: DNA Enzyme Immunoassay.- 3.2.6 Limitation of PCR for the Diagnosis of CMV Infection.- References.- 3.3 HPLC-Analysis of Nucleic Acids.- 3.3.1 Theoretical Background.- 3.3.2 Experimental Procedures.- 3.3.2.1 Benefits of HPLC Analysis of PCR Products.- 3.3.2.2 Drawbacks of HPLC Analysis.- References.- 3.4 Quantitation of Absolute Numbers of mRNA Copies in a cDNA Sample by Competitive PCR.- 3.4.1 Theoretical Background.- 3.4.2 Experimental Procedures.- 3.4.2.1 Generation of an Internal DNA Standard for Competitive PCR.- 3.4.2.2 Purification and Calibration of the Standard Oligonucleotide.- 3.4.2.3 Quantitation of MRP mRNA by Competitive PCR.- 3.4.2.4 Sensitivity and Reproducibility of the Assay.- References.- Acknowledgment.