RNA研究方法（原著第四版）（导读版） 下载 pdf 百度网盘 epub 免费 2025 电子书 mobi 在线

这部成功的实验指导书的第四版，记录了核糖核酸研究的*历程，扩充收集了经检验的、分离和鉴定真核与原核RNA的实验方案。本书将重点放在整体的转录物分析上，包括定量实验和鉴定可变剪接的起始位点，提供了RNA研究中方案选择的清晰概念。本书各章为学生和研究者提供了清晰易掌握的课程，以使实验的产出*化：读者也可选取感兴趣的方案，而不失连续性。书中丰富的流程图、图表和代表性数据在研究项目的计划、实施和评价阶段都特别有用。

前言

1 重温RNA和细胞生物化学

    为什么研究RNA?

    RNA是什么?

        多核苷酸的装配

    RNA的种类

    转录和中心法则

    研究转录的重要动植物模型

    启动子和调控元件

    基因和基因组的组织影响转录

    RNA聚合酶和转录产物

    信使RNA

        典型的mRNA分子的拓扑学

5'帽

引导序列

编码区

尾随序列

多聚A尾

细胞器mRNA

        mRNA的稳定性、转运和转归

        双顺反子mRNA

原核mRNA

mRNA的序列和结构影响翻译

    基因调控的水平

    来自单基因座的mRNA的可变剪接

    反式剪接:mRNA的修复

    小RNA综观

    微小RNA对基因表达的调控

    参考文献

2 分离RNA的策略

    概述

    纯化RNA过程中的目标

    裂解缓冲液配方

        温和的裂解缓冲液

        方案:用低渗裂解分离胞质RNA

        离液性裂解缓冲液

    用含胍的缓冲液分离RNA

    胍-酸-酚抽提技术

        方案:胍-酸-酚抽提

    密度梯度离心

        氯化铯

方案:氯化铯梯度

        三氟乙酸铯

方案:三氟乙酸铯梯度

    同时分离RNA和DNA

        方案:同时分离RNA和DNA

    说说试剂盒

        硅胶的技术

用硅胶柱分离胞质RNA

        亲和介质

    其他方法

方案:用十二烷基硫酸钠和乙酸钾试剂快速分离RNA

        方案:分离原核RNA

        方案:分离酵母RNA

    提纯的RNA的短期和长期保存

    参考文献

3 关于组织的真相

    概述

    组织培养物还是组织?

        细胞培养的优点

        组织样品的优点

    匀浆法

        用Polytron捣碎

        用Dounce匀浆器匀浆

BeadBeaterTM技术

    各种器官和组织的RNA分离的策略

        新鲜组织

        冻存组织

        固定的组织

方案:分离组织RNA的LiCl-尿素法

        方案:从富含类脂样品分离RNA

        与聚核糖体结合的mRNA的提纯

        方案:聚核糖体mRNA的分离

    在野外收集样品

    RNA“清洗”法

    从组织分离RNA的困难解决

    参考文献

4 走向绿色:植物RNA及其分子生物学

    概述

    RNA的分离和植物的怪异点

    植物细胞产生的RNA的种类

    方案:从叶中分离RNA

    方案:从树皮中分离RNA

    方案:从果实中分离RNA

    从其他植物组织分离RNA的策略

    从植物组织分离RNA的困难解决

    参考文献和建议阅读

5 带PolyA尾的RNA的分离

    概述

    加PolyA尾

    利用Poly(A)时的注意事项

        例1

        例2

    选择带PolyA尾的分子

    用于提纯Poly(A)+的磁珠技术

    Oligo(dT)-纤维素柱层析

方案:大量Poly(A)+RNA的提纯

不用柱的快速Poly(A)+提纯

    方案:不用柱的Poly(A)+提纯

    参考文献

6 RNA制品的质量控制

    概述

    质控技术1:紫外分光光度法和光吸收比

        分光光度法

核酸浓度的测定

核酸纯度的测定

        非分光光度法

    质控技术2:RNA的电泳图

        方案

    质控技术3:紫外光投影

        方案

    质控技术4:样品支持RT-PCR的能力

    质控技术5:Northern分析

    质控技术6:样品支持体外翻译的能力

    参考文献

7 棘手的核糖核酸酶

    概述

    核糖核酸酶活力的去除

    潜伏的RNase污染问题

    核糖核酸酶抑制剂的种类

        专一抑制剂

        氧钒核糖核苷络合物

RNasin?

        非专一抑制剂

    仪器和试剂的准备

        焦碳酸二乙酯

        代替剂:灭菌水

        过氧化氢

        氢氧化钠和十二烷基硫酸钠

    控制核酸酶活力用的其他试剂

        盐酸胍

        硫氰酸胍

        十二烷基硫酸钠

        N-十二烷基肌氨酸

        酚∶氯仿∶异戊醇

        8-羟基喹啉

        氯化铯

        三氟乙酸铯

        蛋白酶K

        “RNAlater”(商品名)

    方案:氧钒核糖核苷络合物的合成

    参考文献

8 严谨度:影响核酸结构的条件

    双链分子的种类

    控制严谨度的重要性

        盐对严谨度的影响

        pH对严谨度的影响

        温度对严谨度的影响

        甲酰胺对严谨度的影响

    尿素对严谨度的影响

    参考文献

9 RNA电泳

    概述

    核酸的标准化

        样品制备

        方案:Poly(A)的标准化

    琼脂糖凝胶电泳的RNA变性系统

        甲醛变性

        方案:甲醛变性胶

    尿素变性

        方案:尿素变性

        乙二醛、二甲基亚砜变性

        方案:RNA的乙二醛化和电泳

    用非变性胶跑RNA电泳

    分子量标准

        分子大小标准的恰当应用

        核糖体RNA

    凝胶染色技术

        溴乙锭

        SYBR绿

        SYBR金

        安全SYBR

        “GelStar”染色剂

        银染色

        吖啶橙

        亚甲基蓝

    安全考虑和仪器维护

    次跑琼脂糖电泳:一些提示

        基本词汇表

        要牢记的几点

    参考文献

10 照相记录和影像分析

    概述

    安全

    数字影像分析

        影像格式

        实际的考虑

        适用于任何预算的数字影像分析

        影像分析学习班

        磷屏显像仪

    传统的照相记录方法

        样品显影

        滤光

        将电泳图谱化的几点提示

        照相底片和X光片的内在局限性

    参考文献和建议阅读

11 Northern分析

    概述

    滤膜的选择

        硝酸纤维素膜

        尼龙膜

        聚偏二氟乙烯膜

    处理和滤膜准备

    Northern转移技术

        毛细现像转移

        “TurboBlotter”转移器

        负压转移

        电转移

        碱性转移

        方案:利用被动的毛细扩散转移RNA

方案:用TurboBlotter向下转移RNA

    滤膜转移后的处理

        甲醛变性系统

        乙二醛变性系统

选择1

选择2

    固定化技术

        烘烤

        紫外光交联

        方案:用紫外光把RNA交联在尼龙膜上

    滤膜固定后的处理

    逆Northern分析

    参考文献

12 核酸探针技术

    概述

    探针的分类

    选择标记系统

        同位素标记

尽可能避免分解问题

        非同位素标记

        通用染料Cy3和Cy5

    常用的化学发光方式

        生物素

        洋地黄苷原

        荧光素

        直接酶标记

    DNA探针

        DNA探针的合成

        聚合酶链式反应

        随机引物反应

        缺口转移

        5'末端标记

        3'末端标记

    反义RNA探针

        RNA探针的特征

        RNA探针的合成

体外转录

5'末端标记

3'末端标记

    探针的纯化

    探针的保存

    参考文献

13 核酸杂交的实践

    概述

    影响杂交动力学和双链稳定性的因素

        温度

        离子强度

        pH

        探针长度

        探针浓度

        G+C含量

        错配

        探针复杂度

        粘度

        甲酰胺

        尿素

    杂交温度

        长探针的熔点温度

        寡核苷酸探针的熔点温度

    杂交和Northern分析

        预杂交:滤膜的准备

        方案:长探针预杂交

        探针变性

        杂交

        杂交后的严谨度洗涤

    除去探针和重杂交

        方案:除去探针的通用方法

    参考文献

14 检出的原理

    概述

    放射自显影

        滤膜的处理

        X光片

        安全灯

        曝光时间

        增感屏

        荧光显影

        胶片预闪光

        片匣的种类

        显影和定影

    方案:放射自显影

    非同位素方法

        生物素

        洋地黄苷原

        荧光显影

        直接酶标

        用化学发光检出

化学发光的底物

        显色检出方法

    数字影像系统

    参考文献

15 用抗核酸酶技术定量特定的mRNA

    概述

    基本方法

    探针的选择

    化建议

    潜在困难

    方案:用S1核酸酶分析定量转录物

    方案:用抗核糖核酸酶检验定量转录物

    困难解决

    参考文献

16 核RNA的分析

    概述

    转录速率的检验

        与稳态RNA研究的关系

        核Run-off与核Run-on检验

    方案:核Run-off检验

        细胞的收获和细胞核的制备

        制备易破细胞核的另一方法

        从整块组织制备细胞核的另一方法

        转录物的标记和回收

        靶DNA的制备

        杂交用RNA的制备

        杂交后的洗涤和检出

    方案:核Run-off检验的另一方法

    方案:抗核酸酶检验脉冲标记转录检验

    区分各种RNA聚合酶的活力

    提取核RNA用于稳态分析

    方案:直接分离核RNA

方案:从富含核糖核酸酶的细胞中制备核RNA

    核RNA分析的困难解决

    参考文献

17 cDNA合成

    概述

    cDNA合成——概论

        合成链的考虑

        逆转录酶的选择

        合成第二链的考虑

        经典方法

        基于PCR的方法

    方案:链cDNA合成

    评估cDNA合成的效率

    克隆cDNA

        连接的考虑

        用于连接的酶

    应用

    参考文献

18 逆转录PCR:一种科学和技术形式

    概述

    PCR——概论

    逆转录PCR——一般研究

    PCR样品间沾染的防止

        实验室的设计

        程序方法

        阻挡气溶胶的窍门

        尿嘧啶-N-糖苷酶

    引物设计

        基本规则

Tm的考虑

估计Tm

Tm的精确计算

    网上资源

    多重引物的设计

    化手续

        热稳定聚合酶

        正对照

        负对照

        热启动PCR

        锁定的核酸

        降落PCR

    内对照

    关于转录调控

    PCR产物的分析

    逆转录PCR质量控制的注意点

    验证来自PCR的数据的非PCR方法

    相关技术

        5'RACEPCR

        5'RLM-RACE

        3'RACE-PCR

        巢式PCR

        长距离PCR

        单细胞PCR

        发夹式接头PCR

    猎取可变转录起始位点

    方案:链cDNA合成

    方案:cDNA的PCR扩增

    克隆PCR产物

        方案:把平头PCR产物加A尾

        方案:TA克隆的连接反应

        “拓扑”克隆

    其他扩增方法

RNA的线性扩增(Eberwine过程)

        链替换扩增

        基于核酸序列的扩增(NASBA)

        连接酶链式反应

        参考文献

19 定量PCR技术

    概述

    灵敏度指数

    定量研究

    实时PCR

        实时PCR平台

SYBR绿检验

TaqMan?检验

分子信标

“LUX”牌引物

“蝎子”引物

        熔点曲线分析

    内对照

    外对照

    对照反应的设置方式

    负对照的考虑

    竞争PCR:关键的考虑

    竞争PCR:所包括的主要步骤

    另一方法:非实时竞争PCR

        方案:竞争PCR

非同源竞争物的合成

第1链cDNA的合成

竞争PCR(一级扩增)

竞争PCR(二级扩增)

        影像分析的考虑

        定量PCR技术的困难解决

    参考文献

20 功能基因组学和转录物总体分析

    概述

    功能基因组学的定义

    功能基因组学研究方法的重要性

    常用的功能基因组学研究方法

    功能基因组学和经典分子生物学

21 基因表达的高通量分析

    概述

    什么是微阵列?

    什么是热图*?

    微阵列能做什么?

    微阵列不能做什么?

    微阵列分析的主要步骤

    参考RNA

    宏阵列是什么?

    应用

    参考文献

22 整体分析基因表达的非阵列方法

    概述

    基本问题

        消减方法

抑制性消减杂交(SSH)

困难解决

    非消减方法

        mRNA差别显示

各种方案

    参考文献

23 RNA干扰:Dicer酶在RISC复合物中起作用*

    概述

    RNAi的基本名词

    RNA干扰——它如何工作

        siRNA方法

        shRNA方法

        将siRNA导入哺乳动物细胞的方法

        miRNA

    siRNA的有效设计

    RNAi和转录物可变剪切

        体外和体内问题

    RNAi的确认

        RT-PCR方法

        Northern杂交分析

        Western杂交分析

    应用

    参考文献

24 基因组、转录组、蛋白质组和生物信息学

    概述

    基本名词

    基因组和基因组学

    转录组和转录组学

    蛋白质组和蛋白质组学

    生物信息学

    搜索基因——BLAST一下!

    参考文献

25 RNA一例

    一个典型实验?

    灵敏度问题

    下一步做什么

    到哪儿去求助

尾声

    几粒智慧之珠

附录A 保留完整准确的记录

附录B 把质量转换为摩尔

附录C 对分子生物学家有用的储存液

附录D 酚的制备

附录E 溴乙锭和SYBR绿溶液的弃去

附录F 用DNase I从RNA样品中除去DNA

附录G 用RNase保温从DNA样品中除去RNA

附录H 甲酰胺、甲醛和乙二醛的去离子

附录I 硅化离心管和玻璃器具

附录J 贴壁细胞的胰酶处理方案

附录K 盐析法分离高分子量DNA

附录L 电泳:原理、参数和安全

附录M 聚丙烯酰胺凝胶电泳

附录N 点杂交分析

附录O 离心是分子生物学家的主流工具

附录P 仪器和试剂供应商及服务商选登

附录Q 有用的国际单位制单位

附录R 常用缩写

附录S 商标摘录

术语表

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1 RNA and the Cellular

Biochemistry Revisited

Why study RNA? 1

What is RNA? 2

Assembly of polynucleotides 5

Types of RNA 8

Transcription and the central dogma 10

Important plant and animal models for

studying transcription 11

Promoters and regulatory elements 12

Gene and genome organization affect

transcription 14

RNA polymerases and the products

of transcription 18

Messenger RNA 22

Topology of a typical mRNA

molecule 23

5  Cap 23

Leader sequence 25

Coding region 25

Trailer sequence 26

Poly(A)tail 26

Organellar mRNAs 28

mRNA stability,transport,and

turnover 29

Bicistronic mRNAs 30

Prokaryotic mRNAs 30

mRNA sequence and structure affect

translation 31

Levels of gene regulation 32

Alternative splicing of mRNA from a single

genetic locus 34

Trans-splicing: mRNA repair 35

Overview of small RNAs 36

miRNA regulation of gene expression 37

References 38

Why study RNA?

All cell and tissue functions are ultimately governed by gene

expression.

Consequently,the reasons for electing to study the modulation of

RNA as at

least one parameter of the cellular biochemistry may be as diverse

as the intracellular

RNA population itself.Generally speaking,the characterization of

RNA

is almost always related to transcription(i.e.,gene

expression)questions being

asked in the context of a particular scientific inquiry,and most

often revolves

around measuring the dynamic abundance level of one or several

transcripts.

The goals in any experimental design involving RNA generally

revolve around

one or more fundamental themes,including but not limited to the

following:

1.Measurement of the steady-state 1 abundance of cellular

transcripts.This is the most

commonly studied parameter of gene expression primarily because of

the ease with

which RNA can be isolated.Quantitative and qualitative profiles of

a population

of RNAs can then be generated by contemporary methods,such as

PCR-based

approaches,or through the use of time-honored techniques such as

Northern analysis

or nuclease protection.

2.Measurement of the rate of transcription of gene sequences or the

pathways of

RNA processing.This may be deduced,at least in part,by the nuclear

run-on assay

in which radiolabeled ribonucleotide precursors are incorporated

into nascent transcripts,

which occurs in direct proportion to the abundance of each RNA

being

transcribed.When used in conjunction with Northern analysis or

other methods

that examine steady-state RNA levels,the regulation of genes can

often be assigned

as transcriptional or due to posttranscriptional events.

3.Identification of the transcription start

site(TSS).Historically,mapping of RNA

molecules,including the 5'end,the 3'end,and the size and location

of introns,

was accomplished via the nuclease protection assay,as described in

Chapter 15.

Now,however,transcript mapping is now almost always performed by

rapid amplification

of cDNA ends(5'- and 3'RACE; see Chapter 18).Because a single

genetic

locus can produce multiple RNAs,each with a different TSS,and often

in a tissuespecific

manner,this type of analysis is extremely useful and in widespread

use.

4.Synthesis of complementary DNA(cDNA).Unstable,single-stranded

mRNA can

serve as the template for the in vitro synthesis of very stable

single- or double-stranded

cDNA molecules.Among the more common reasons for performing cDNA

synthesis

is the amplification of the sequence by PCR,often for some “

quantitative purpose ” ,

for transcript mapping purposes,for direct ligation into a vector

for sequencing or for

expression of the encoded protein,for the physical separation of

two or more cDNA

species,or for the synthesis of an entire cDNA library 2 which can

be propagated for

long-term storage and analysis.In so doing,one constructs a

permanent record of the

cellular biochemistry at the moment of cell lysis or tissue

disruption.Historically,the

synthesis of highly representative cDNA is one of the most

important,yet technically

challenging,methodologies in the molecular biology

laboratory.

5.In vitro translation of purified messenger RNA(mRNA).The

resulting polypeptide

may be further characterized by immunoprecipitation or Western

analysis.Cell-free

translation represents at least one method for the identification

of specific transcripts:

by providing the raw materials needed to support translation,one is

able to demonstrate

that a transcript of putative identity is able to support the

synthesis of the

cognate peptide.For example,this approach could be used to

demonstrate that two

transcripts from the same genetic locus with alternative

transcription start sites are,

in fact,able to direct the synthesis of identical or closely

related proteins.In applications

such as rational drug design,in vitro translation is also helpful

because understanding

the three dimensional architecture of a protein,and its wild type

or mutated

function(s),may suggest novel applications in the area of

functional proteomics.

What is RNA?

RNA is a long,unbranched polymer of ribonucleoside monophosphate

moieties

joined together by phosphodiester linkages,and both eukaryotic

and

prokaryotic RNAs are essentially single-stranded molecules.The

unassembled

monomers of both RNA and DNA are called nucleotides.These

building

blocks consist of three key components: a pentose(five-carbon

sugar),at least

one phosphate group(nucleotides may contain as many as three

phosphate

groups),and a nitrogenous base( Fig.1.1 ).A nitrogenous base joined

to a pentose

sugar is known as a nucleoside.When a phosphate group is

added,the

composite,a phosphate ester of the nucleoside,is referred to as a

nucleotide.

Base+sugar=nucleoside

Nucleoside+phosphate=nucleotide

The key chemical difference between RNA and DNA is the presence the

fivecarbon

sugar ribose,in which a hydroxyl group(-OH)is joined to the

2'carbon

of the ribose sugar whereas the absence of this-OH group in DNA

is

the underlying basis of the name of the sugar deoxyribose.In

addition,one

finds the base uracil in RNA,substituted in DNA by the closely

pyrimidine

thymine 3 ,though it is possible to find nucleotides containing

uracil in DNA in

certain situations.More precisely,RNA is assembled from

ribonucleotide precursors

and DNA is assembled from deoxyribonucleotide

precursors.Hence,

RNA is so-named because of the ribose sugar it contains,just as DNA

is named

from its constituent 2'-deoxyribose sugar.The principal features

and essential

nomenclature of these building blocks are summarized in Tables 1.1

and 1.2 .

Nitrogenous bases and the pentose sugar components of nucleosides

are

both cyclic.To avoid confusion when referring to the constituent

atoms of the

sugar or the base that constitute a particular nucleotide,a special

numbering

system has been devised.By convention,the numbering system for the

carbon

and nitrogen atoms that make up the bases is 1,2,3,and so

forth,while the

numbering system for the constituent carbon atoms of the

sugar(ribose or

deoxyribose)is 1',2',3',4',and 5'.

The ribonucleoside triphosphates may be collectively(and sometimes

singularly)

referred to as NTP; in various molecular biology protocols the

symbol

NTP refers to an equimolar cocktail of ATP,CTP,GTP,and

UTP.Similarly,

the deoxy- form of a nucleotide is denoted by the placement of a

lower case

“d” preceding the nucleotide triphosphate,as in dATP,dCTP,dGTP,and

dTTP,

and the symbol dNTP refers to an equimolar cocktail of the four

deoxynucleoside

triphosphates in protocols capable of supporting the de novo

synthesis of

cDNA or PCR products.It is the triphosphate form of each of these

nucleotides

that is recognized by RNA polymerase and DNA

polymerase,respectively,

thereby serving as precursors in nucleic acid synthesis.The

phosphate nearest

the nucleoside moiety,that is,closest to the sugar,is known as the

α phosphate,

followed by the β phosphate,followed by the γ phosphate,which is

furthest

from the nucleoside.During nucleic acid polymerization,the β and

γ

phosphates are cleaved from the nucleotide,and the resulting

single-phosphate

nucleotide is then incorporated into the nascent polynucleotide

chain.

Assembly of polynucleotides

The synthesis of RNA is mediated by the activity of enzymes known

as RNA

polymerases while DNA is synthesized via the enzymatic activity of

DNA

polymerases.Thus,any enzyme with an associated polymerase activity

is capable

of synthesizing nucleic acid molecules from nucleotide precursors.A

nucleic

acid molecule is the result of linking nucleotides together by

phosphodiester

bonds.The formation of these bonds involves the hydrophilic attack

by the 3'

hydroxyl group of the last nucleotide added to the nascent

polynucleotide on

the 5'phosphate group of the incoming nucleotide.

Assuming that the intracellular biochemistry can support initiation

and elongation

of RNA molecules,there are two fundamental requirements that

must

be fulfilled and maintained in vivo(and in vitro )to support

continued nucleic

acid polymerization:

1.There must be a template strand to direct the polymerase-mediated

insertion of the

correct(complementary)nucleotide into the nascent chain.This occurs

predictably,

according to the conventions set down in Chargaff’s Rule( Zamenhof

et al .,1952 ),

which succinctly states that adenine ordinarily base-pairs with

thymine or uracil

through the formation of two hydrogen bonds(A::T,A::U)and that

guanine ordinarily

base-pairs to cytosine through three hydrogen bonds(G:::C).

2.For initiation and elongation,there must be a free 3'-OH to which

the next nucleotide

in the chain can be joined via a phosphodiester linkage.Thus,the

entire process

of transcription in vivo and in vitro requires some type of primer

manifesting the

requisite 3'-OH.The same is true for DNA synthesis 4 .Most of the

enzymes used

in molecular cloning that exhibit polymerase activity have nearly

identical template

and 3'-OH primer requirements.

Polynucleotide elongation involves the incorporation of a

nucleoside monophosphate,

and the formation of this linkage is accompanied by the release

of

pyrophosphate.The synthesis of nucleic acids,both RNA and

DNA,always pro -

ceeds from the 5'direction toward the 3'direction,resulting in a

polynucleotide

that manifests a consistent pattern of 5'→ 3'linkages between

adjacent

nucleotides.Elongation is frequently referred to as the 5'→

3'polymerase activity

associated with the enzyme.

Upon completion,nucleic acid molecules are assembled in such a way

that:

1.The ends of the molecule are structurally different from one

another.The first

nucleotide of the molecule has an uninvolved

5'(tri)phosphate,constituting the socalled

5'end of the molecule.The last nucleotide that was added manifests

a free 3'

hydroxyl group,and this is known as the 3'end of the

molecule.

2.The backbone of the molecule consists of an alternating series of

sugar and phosphate

groups.Known as the phosphodiester backbone,or simply the

backbone,of

the molecule,it imparts a net negative charge to the molecule by

virtue of its constituent

phosphate groups.

3.The base associated with each nucleotide protrudes away from the

backbone of the molecule.

This stereochemistry makes the bases very accessible for hydrogen

bonding(base

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书籍介绍

《RNA研究方法(原著第4版)(导读版)(英文版)》的目录和前言已经译成中文，正文部分保留英文原版。另附中国科学院上海生命科学研究院生物化学与细胞生物学研究所刘定干研究员所作精彩导读一篇。

这部成功的实验指导书的第四版，记录了核糖核酸研究的最新历程，扩充收集了经检验的、分离和鉴定真核与原核RNA的实验方案。《RNA研究方法(原著第4版)(英文版)》将重点放在整体的转录物分析上，包括定量实验和鉴定可变剪接的起始位点，提供了RNA研究中方案选择的清晰概念。《RNA研究方法(原著第4版)(导读版)(英文版)》各章为学生和研究者提供了清晰易掌握的课程，以使实验的产出最优化；读者也可选取感兴趣的方案，而不失连续性。书中丰富的流程图、图表和代表性数据在研究项目的计划、实施和评价阶段都特别有用。