Gene Expression - Relative Quantification-2
Epigenetics and Gene Expression
Epigenetics and Gene ExpressionEpigenetics and gene expression are two interconnected fields of study that have been gaining a lot of attention in recent years. Epigenetics refers to the study of changes in gene expression that occur without any changes to the underlying DNA sequence. Gene expression, on the other hand, refers to the process by which genetic information is used to produce proteins and other molecules that are essential for life. In this response, I will explore the relationship between epigenetics and gene expression from multiple perspectives.From a scientific perspective, epigenetics and gene expression are intimately linked. Epigenetic modifications, such as DNA methylation and histone modification, can have a profound impact on gene expression. For example, DNA methylation can prevent the binding of transcription factors to DNA, which in turn can prevent the transcription of specific genes. Similarly, histone modifications can alter the structure of chromatin, making it more or less accessible to transcription factors. These modifications can be passed down from one generation to the next, and can even be influenced by environmental factors such as diet and stress.From a medical perspective, the study of epigenetics and gene expression has the potential to revolutionize our understanding of disease. Many diseases, such as cancer and Alzheimer's, are thought to be caused by changes in gene expression. By understanding the epigenetic mechanisms that regulate gene expression, we may be able to develop new treatments for these diseases. For example, drugs that target specific epigenetic modifications may be able to restore normal gene expression patterns in cancer cells, leading to their death. Similarly, drugs that target epigenetic modifications associated with Alzheimer's may be able to slow or even reverse the progression of the disease.From a social perspective, the study of epigenetics and gene expression raises important ethical questions. For example, if epigenetic modifications can be passed down from one generation to the next, what are the implications for future generations? Could environmental factors such as pollution or stress have long-lasting effects on the health of future generations? Similarly, if epigenetic modifications can be influenced by diet andlifestyle, what are the implications for public health policy? Should governments be investing in programs that promote healthy lifestyles in order to prevent epigenetic modifications that could lead to disease?From a personal perspective, the study of epigenetics and gene expression has the potential to change the way we think about ourselves and our place in the world. For example, if epigenetic modifications can be influenced by environmental factors such as diet and stress, then our health may be more within our control than we previously thought. This could be empowering for individuals who are looking to take control of their health. Similarly, if epigenetic modifications can be passed down from one generation to the next, then our actions today may have long-lasting effects on the health of future generations. This could be a sobering realization for individuals who are concerned about the impact of their actions on the world around them.From a philosophical perspective, the study of epigenetics and gene expression raises important questions about the nature of life and the role of genetics in shaping our identities. For example, if epigenetic modifications can have a profound impact on gene expression, then what does this say about the role of genetics in shaping our identities? Are we more than the sum of our genes, or are we simply the products of our genetic makeup and the epigenetic modifications that occur throughout our lives? Similarly, if epigenetic modifications can be influenced by environmental factors such as diet and stress, then what does this say about the nature of life itself? Are we simply passive recipients of environmental influences, or do we have the power to shape our own destinies?In conclusion, the study of epigenetics and gene expression is a complex and multifaceted field that has the potential to revolutionize our understanding of disease, raise important ethical questions, change the way we think about ourselves and our place in the world, and challenge our fundamental assumptions about the nature of life and the role of genetics in shaping our identities. As scientists, medical professionals, policymakers, and individuals, it is our responsibility to engage with these questions in a thoughtful and nuanced way, in order to ensure that we are making the most of this exciting and rapidly evolving field of study.。
differential gene expression analysis
differential gene expression analysisDifferential gene expression analysis（差异基因表达分析）是一种研究基因表达模式在不同条件或不同组织样本之间差异的方法。
通过比较不同条件或组织样本的基因表达谱，可以发现哪些基因的表达水平发生了显著变化，从而了解这些基因在生物学过程或疾病发展中的作用。
在进行差异基因表达分析之前，通常需要对基因表达数据进行标准化处理，以确保不同样本之间的数据具有可比性。
然后，使用统计方法比较不同条件或组织样本的基因表达谱，筛选出表达差异显著的基因。
这些差异基因可能涉及不同的生物学过程、信号通路或疾病过程，具有重要的生物学意义。
差异基因表达分析在许多领域都有应用，如生物学、医学和农业等。
例如，在生物学研究中，差异基因表达分析可以用于研究生物生长发育过程中的基因表达变化；在医学研究中，差异基因表达分析可以用于研究疾病发生发展过程中的基因表达变化，从而发现潜在的治疗靶点或药物。
总之，差异基因表达分析是一种强大的工具，可以帮助我们深入了解基因表达模式的变化，揭示生物学过程和疾病机制，为药物研发和疾病治疗提供重要的线索和依据。
在差异基因表达分析中，数据标准化处理是非常重要的一步，其目的是消除不同样本或实验条件之间的系统误差，使数据具有可比性。
以下是一些常用的数据标准化处理方法：1.归一化：将每个样本的基因表达量转换为相对表达量，使不同样本之间具有可比性。
常见的归一化方法包括：•截尾值归一化：将表达量低于某一阈值的基因去除，或将其表达量设为0。
•最大值归一化：将每个样本的表达量除以该样本中表达量的最大值，使所有样本的表达量都在0-1之间。
•平均值归一化：将每个样本的表达量减去该样本表达量的平均值，使所有样本的表达量都为0。
1.批间归一化：由于实验过程中可能存在的批次效应，需要对不同批次的样本进行归一化处理，使它们之间具有可比性。
分子遗传学常用词汇
分子遗传学常用词汇词汇医学英语专业英语医学术语分子遗传学【字体：小大】腺嘌呤Adenine（A）：一种碱基，和胸腺嘧啶T结合成碱基对。
等位基因（Alleles）：同一个基因座位上的多种表现形式。
一般控制同一个性状，比如眼睛的颜色等。
氨基酸（Amino Acid）：共有20种氨基酸组成了生物体中所有的蛋白质。
蛋白质的氨基酸序列和由遗传密码决定。
扩增（Amplification）：对某种特定DNA片段拷贝数目增加的方法，有体内扩增和体外扩增两种。
（参见克隆和PCR技术）克隆矩阵（Arrayed Library）：一些重要的重组体的克隆（以噬菌粒，YAC或者其他作载体），这些重组体放在试管中，排成一个二维矩阵。
这种克隆矩阵有很多应用，比如筛选特定的基因和片段，以及物理图谱绘制等。
从每种克隆得到的遗传连锁信息和物理图谱信息都输入到关系数据库中。
自显影技术（Autoradiography）：使用X光片来显示使用放射性元素标记的DNA片段的位置，常用在使用凝胶将DNA片段按照片段大小分离之后，显示各个DNA片段的位置。
常染色体（Autosome）：和性别决定无关的染色体。
人是双倍体动物，每个体细胞中都含有46条染色体，其中22对是常染色体，一对是性染色体（XX或者XY）。
噬菌体（Bacteriophage）：参见phage碱基对（Base Pair，bp）：两个碱基（A和T，或者C和G）之间靠氢键结合在一起，形成一个碱基对。
DNA的两条链就是靠碱基对之间的氢键连接在一起，形成双螺旋结构。
碱基序列（Base sequence）：DNA分子中碱基的排列顺序。
碱基序列分析（Base Sequence Analysis）：分析出DNA分子中碱基序列的方法（这种方法有时能够全自动化）cDNA：参见互补DNA厘摩（cM）：一种度量重组概率的单位。
在生殖细胞形成的减数分裂过程中，常常会发生同源染色体之间的交叉现象，如果两个标记之间发生交叉的概率为1％，那么它们之间的距离就定义为1cM.对人类来说，1cM大致相当于1Mbp.着丝点（Centromere）：在细胞的有丝分裂过程中，从细胞的两端发出纺锤丝，连接在染色体的着丝点上，将染色体拉向细胞的两级。
多重基因表达
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逆转录多聚酶链反应(RT-PCR)
RT-PCR是将RNA的反转录（RT） 和cDNA的聚合酶链式扩增（PCR） 相结合的技术。 首先经反转录酶的作用从RNA合 成 cDNA，再以cDNA为模板，扩 增合成目的片段。 优点: 1 灵敏、用途广泛：可用于检测 细胞中基因表达水平，细胞中 RNA病毒的含量和直接克隆 特定 基因的cDNA序列。 2作为模板的RNA可以是总RNA、 mRNA或体外转录的RNA产物。 缺点：通量小，每次1-2个基因
There are no 30,000 gene diseases No compound impacts 30,000 genes
Gene Expression Trend
10,000
Discovery Phase Whole genome scan Modest sample set ID Biomarkers Signature Phase Focused gene set (4-100)
# of Samples
基因芯片(gene chip)
优势:高通量 缺点:成本高、数据量大
QPCR原理
Wild-Type DNA
5’...GCGGACCGCG..
After Bisulfite Treatment: Unmethylated DNA 5’...GUGGAUUGUG..
Methylated DNA
5’...GCGGAUCGCG..
高通量 SNP 筛查
SNP Amplifluor™ Primer (UniPrimer™)
在定量PCR开始前增加50°C的保温步骤， UNG酶即可将已有的PCR产物降解破坏，防止可能造成的污染
Block temperature [°C]
仪器热启动
100
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80
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60
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超快的升温速度 +
极大缩短初始升温所需时间
40
20
• Innovative tecThimniecal feature for device-supported HotStart PCR
医学方面应用前景更是令人鼓舞 •极微量的基因表达定量 •病原体检测
绝通对常定用量标准品作为外参照制作标准曲线.
注意保持与目的基因的同源性
Unknown
标准曲线的制作
什么是阈值
基线信号的标准偏差 x 10
基线
标
阈
准
值
偏
差
基线阈值CT值
基线
阈 值
CT值
标准曲线方法
20- ••
1918-
CT
171615-
PCR Quencher
Real Time PCR 技术的诞生
• 1992年，Higuchi R等第一个报导了实时定量PCR技 术
Higuchi R, Fockler C, etal. Kinetic PCR analysis: real-time monitoring of DNA amplification reactions. Biotechnology,
相对荧光定量PCR的常用方法和注意事项
相对定量方法实际操作（常用方法）parative Delta-delta Ct法定量流程（RG6000软件设置）1).先对样品中的目的基因与看家基因分别做标准曲线，通过标准曲线确定两个基因的扩增效率是否一致或接近；将扩增效率优化为一致。
2).同一样品分别进行看家基因和目的基因的扩增，分列在两页中P1 P2相同的样品在两页里命名成相同的名称，并定义为unknown分别分析P1和P2页选delta-delta Ct选项依次填入，并定义对照样品公式：Comparative Delta-delta Ct 法的特点、注意事项及实际应用1). Comparative Delta-delta Ct 法是很常用的一种相对定量方法，其最大特点是，当优化的体系已经建立后，在每次实验中无需再对看家基因和目的基因做标准曲线，而只需对待测样品分别进行PCR 扩增即可。
2). 其缺点是，每次实验都默认目的基因和看家基因的扩增效率一致，而并非真实扩增情况的反映，这里势必存在一定的误差。
3). Comparative Delta-delta Ct 法展开定量实验前，在预实验中，必需对目的基因和看家基因做两组标准曲线。
Rotor-Gene 的软件会自动给出两组标准曲线的R 值、扩增效率等信息，如果两组标准曲线的斜率，即M 值的差小于0.1，那么后续实验中就可以用Comparative Delta-delta Ct 法进行相对完成分析F=2—待检样品看家基因平均Ct 值对照组目的基因平均Ct 值 对照组看家基因平均Ct 值— 待检样品目的基因平均Ct 值 ——定量分析。
反之，如果M差值大于0.1，就无法用该方法进行相对定量分析。
此时的解决方法有两种，一是优化实验，使两组标准曲线的斜率差值小于0.1，二是换用其它的相对定量方法。
应用实例：如上图，将标准品进行梯度稀释后，分别对目的基因（Gene of Interest）和看家基因（Housekeeper Gene）做标准曲线。
Relative quantification of 40 nucleic acid sequences by multiplex ligationdependent probe amplificat
©2002Oxford University Press Nucleic Acids Research,2002,Vol.30,No.12e57 Relative quantification of40nucleic acid sequences by multiplex ligation-dependent probe amplificationJan P.Schouten*,Cathal J.McElgunn,Raymond Waaijer,Danny Zwijnenburg,Filip Diepvens and Gerard Pals1MRC-Holland,Hudsonstraat68,1057SN Amsterdam,The Netherlands and1Department of Clinical Genetics,Free University of Amsterdam,v.d.Boechorststraat7,1081BT Amsterdam,The NetherlandsReceived February22,2002;Revised and Accepted April20,2002ABSTRACTWe describe a new method for relative quantification of40different DNA sequences in an easy to perform reaction requiring only20ng of human DNA.Applica-tions shown of this multiplex ligation-dependent probe amplification(MLPA)technique include the detection of exon deletions and duplications in the human BRCA1,MSH2and MLH1genes,detection of trisomies such as Down’s syndrome,characterisa-tion of chromosomal aberrations in cell lines and tumour samples and SNP/mutation detection.Relative quantification of mRNAs by MLPA will be described elsewhere.In MLPA,not sample nucleic acids but probes added to the samples are amplified and quantified.Amplification of probes by PCR depends on the presence of probe target sequences in the sample.Each probe consists of two oligonucleotides, one synthetic and one M13derived,that hybridise to adjacent sites of the target sequence.Such hybrid-ised probe oligonucleotides are ligated,permitting subsequent amplification.All ligated probes have identical end sequences,permitting simultaneous PCR amplification using only one primer pair.Each probe gives rise to an amplification product of unique size between130and480bp.Probe target sequences are small(50–70nt).The prerequisite of a ligation reaction provides the opportunity to discrim-inate single nucleotide differences. INTRODUCTIONChanges in copy number of certain specific chromosomal sequences are frequently implicated in the cause of,or dispos-ition to,human diseases and syndromes.Such changes include the presence of an extra copy of a complete chromosome as in Down’s syndrome,deletions of up to several million base pairs as in DiGeorge syndrome and deletions or duplications of smaller chromosomal fragments such as a single exon.For instance,>60%of Duchenne and Becker muscular dystrophy cases are due to deletions or duplications of one or more exons of the DMD gene(1).Deletion or duplication of one or more exons of the BRCA1or the MLH1/MSH2genes predispose the carrier to breast and colon cancer(2,3),respectively.In the same way,copy number changes of specific chromosomal regions are very common in tumours and are an important factor influencing gene expression(4).Analysis of these copy number changes is important for treatment as exemplified by the introduction of ERBB2-specific antibodies for the treatment of breast cancer patients with ERBB2gene amplification(5).At present,many different techniques are used for the detection of copy number changes of chromosomal sequences including standard chromosome analysis,comparative genomic hybridisation(CGH)(6),fluorescent in situ hybridisation (FISH)(7),BAC arrays(8),Southern blots and loss of hetero-zygosity(LOH)(9)assays.Most of these techniques are not able to detect deletions or duplications of single exons.Besides they are time consuming,difficult to implement as multiplex assays(FISH,LOH)or require large amounts of sample DNA (Southern blots).The conventional methods for mutation detection that are based on PCR amplification of single exons from genomic DNA will not detect most exon deletions and duplications as a normal allele is also present.Whilst oligo-nucleotide and cDNA microarrays are still insufficiently sensitive and reproducible to detect a deletion or duplication of one copy of a small chromosomal sequence like a single exon,real-time PCR provides the possibility to detect several fold amplifica-tion of chromosomal sequences;however,its use in a multi-plex assay is severely limited by spectral overlap of the fluorescent dyes used.In addition,the presence of multiple primer pairs in a multiplex reaction reduces the robustness of PCRs and the reliability of the quantification.Yet the use of multiplex techniques is essential for the routine detection of exon deletions and duplications in genes like BRCA1,with24 exons,or DMD,having79exons.Analysis of the large variety of aberrations found in tumours also requires multiplex nucleic acid analysis methods that need to be very sensitive in view of the often restrictive amounts of sample available.Nucleic acid fingerprinting techniques,such as amplified fragment length polymorphism(AFLP)(10)and differential mRNA display(11),have clearly demonstrated that PCR can be used for simultaneous reproducible amplification of many DNA fragments in one reaction,provided that only a single primer set is used for amplification of all these fragments. AFLP can be used to determine the relative copy number of*To whom correspondence should be addressed.Tel:+31204447218;Fax:+31206891149;Email:schouten@more than50random sequences in a single reaction.Multiplex amplifiable probe hybridisation(MAPH)(12)is a similar method in which not random fragments but40different specific target sequences are detected and quantified.MAPH uses oligonucleotide probes that hybridise to specific nucleic acid sequences.Each hybridised probe can be simultaneously amplified with the use of a single primer pair and yields an amplification product of unique size.The copy number of target sequences is reflected in the relative intensities of the MAPH probe amplification products(12).However,like Southern blotting,MAPH requires immobilisation of sample nucleic acids and tedious washing of unbound(amplifiable) probes making it difficult to implement in a routine diag-nostic setting.Here,we introduce a related technique named multiplex ligation-dependent probe amplification(MLPA) that is more sensitive and easier to use.As in MAPH,added oligonucleotide probes rather than sample nucleic acids are amplified.However,immobilisation of sample nucleic acids and removal of excess probes is not necessary.MATERIALS AND METHODSProbe preparationEach MLPA probe consists of one short synthetic oligonucle-otide and one,phage M13-derived,long probe oligonucleotide. The short synthetic oligonucleotide of each probe contains a target-specific sequence(21–30nt)at the3′end and a common 19nt sequence,identical to the labelled PCR primer,at the 5′end.Preparation of the long MLPA probe oligonucleotide is outlined in Figure1.For the preparation of each long MLPA probe,a target sequence-specific oligonucleotide of25–43nt is cloned in one of the M13-derived SALSA vectors.Each clone obtained is used to infect a1-l culture of Escherichia coli strain TG1.Single stranded DNA is purified by polyethyleneglycol precipitation of phage particles,heat disruption of the virus and cetyl-trimethyl-ammonium bromide precipitation of the DNA. This single stranded DNA is made partially double stranded at the Eco RV and Bsm I sites by the annealing of two short oligo-nucleotides.Digestion by Eco RV and Bsm I results in the formation of a large7200nt M13fragment and the MLPA probe oligonucleotide(80–420nt).This probe oligonucleotide contains the25–43nt target-specific sequence at the5′phos-phorylated end,a36nt sequence that contains the complement of the unlabelled PCR primer and is common to all probes at the3′end,and a stuffer sequence of variable length in between. It is not necessary to remove the large7000nt M13fragment before use.Melting temperatures of all probe target-specific sequences are>65°C at100mM NaCl.For the design of the hybridising probe parts,sequence information available from the public databases was used.Except for the labelled PCR primers,synthetic oligonucleotides were not purified.MLPA analysisDNA samples were diluted with TE to5µl and were heated at 98°C for5min in200µl tubes in a thermocycler with a heated lid(Biometra Uno II).After addition of1.5µl salt solution (1.5M KCl,300mM Tris–HCl pH8.5,1mM EDTA)mixed with1.5µl probe mix(1–4fmol of each synthetic probe oligo-nucleotide and each M13-derived oligonucleotide in TE), samples were heated for1min at95°C and then incubated for 16h at60°C.Ligation of annealed oligonucleotides was performed by diluting the samples to40µl with dilution buffer (2.6mM MgCl2,5mM Tris–HCl pH8.5,0.013%non-ionic detergents,0.2mM NAD)containing1U Ligase-65enzyme, and incubation for15min at54°C.The ligase enzyme was inactivated by heating at98°C for5min and ligation products were amplified by PCR.For most experiments,10µl of the ligation reaction was added to30µl PCR buffer.While at 60°C,10µl of a buffered solution containing the PCR primers (10pmol),dNTPs(2.5nmol)and 2.5U Taq polymerase (Promega)or SALSA polymerase(MRC-Holland)were added.Alternatively,the10µl solution containing PCR primers,dNTPs and polymerase was added to the complete MLPA reactions while at60°C.PCR was for33cycles(30s at 95°C,30s at60°C and1min at72°C).Samples amplified with one unlabelled and one IRD-800labelled primer(LICOR) were analysed on a6.5%acrylamide slab gel in a LICOR IR2 system.Samples amplified with one unlabelled and one D4-labelled primer(Research Genetics)were analysed on a Beckman CEQ2000capillary electrophoresis system.MLPA reactions resulting in only10–12amplification products were analysed by electrophoresis on ethidium bromide stained agarose gels.Two-fold differences in relative amounts of target sequences could be scored by visual examination.The sequence of the labelled primer is5′-GGGTTC-CCTAAGGGTTGGA-3′and that of the unlabelled primer is 5′-GTGCCAGCAAGATCCAATCTAGA-3′.The exact gene and sequence recognised by each probe used in this article can be found at .RESULTSAn outline of the MLPA reaction is shown in Figure2.DNA (20–500ng)is denatured and fragmented by a5min heat treat-ment at98°C.MLPA probes are added and allowed to hybridise for16h at60°C in a thermocycler with a heated lid. Dilution buffer including the ligase enzyme is added and ligation is allowed to proceed for15min at54°C.After heat inactivation of the ligase and addition of PCR primers,dNTPs and polymerase,PCR amplification of the ligation products is started.One PCR primer is fluorescently or isotopically labelled.Amplification products are detected and quantified by capillary electrophoresis(see Figs4–8)or traditional gel electrophoresis(Fig.3).Preparation and design of MLPA probesLigation-dependent PCR has been described previously (13–15).Several modifications were made to make the technique simple to perform,more reproducible and sensitive, and suitable for true multiplex analysis.Each MLPA probe consists of two oligonucleotides that can be ligated to each other when hybridised to a target sequence. All ligated probes have identical sequences at their5′and3′ends,permitting simultaneous amplification in a PCR containing only one primer pair.Each probe gives rise to an amplification product of unique size between130and480bp.One of the two oligonucleotides of each MLPA probe is chemically synthe-sised and has a common sequence used for PCR amplification at the5′end and a target-specific sequence at the3′end.Each second probe oligonucleotide contains a sequence of25–43nt at the5′end that is able to hybridise to the target sequenceimmediately adjacent to the first probe oligonucleotide,a common sequence used for PCR amplification at the 3′end,and a stuffer sequence of 19–370nt in between.Chemically synthesised oligonucleotides of this size (80–440nt)are not commercially available in the quality needed for MLPA.We therefore use single stranded DNA from M13clones containing small target-specific sequences for the preparation of these oligonucleotides.The M13DNA is made partially double stranded by the annealing of complementary oligonu-cleotides and is digested by two restriction endonucleases (Fig.1).One of these enzymes cuts the DNA outside its recognition sequence,resulting in a 5′phosphorylated end that is perfectly complementary to the target sequence.Most probe mixes made contain 35–42probes with length differences between consec-utive amplification products of 6or 9bp.All probes used in a specific MLPA probe mix are made in a different M13-derived vector and have different stuffer and hybridising sequences.Amplification products of different probes have common sequences only at their ends in order to prevent heteroduplex formation during later stages of the amplification reaction when competition between duplex formation and PCR primer annealing takes place.We prepared a set of 118different M13-derived MLPA vectors,each containing a stuffer sequence of different length and sequence.Targetsequence-specificFigure 1.Preparation of the long M13-derived MLPA probe oligonucleotides.The basic MLPA vector M214was derived from M13mp18(24)by destroying the single Bsm I site and replacement of the polylinker site by a synthetic oligonucleotide containing,from 5′to 3′,a new Bsm I site,an Sph I and Xba I site,a sequence complementary to one of the two SALSA PCR primer sequences and an Eco RV site.A collection of 118different SALSA vectors was prepared by insertion of stuffer fragments in the Sph I and Xba I sites.Each stuffer fragment has a different sequence and length (19–370bp,3bp increments).Stuffer fragments up to 55nt were synthetic;longer fragments were made by PCR amplification of T7phage sequences.For each MLPA probe,a synthetic 30–43nt long oligonucleotide containing the hybridising sequence is cloned in the Sph I and Bsm I sites of one of the SALSA vectors.Single stranded DNA is prepared from the clone obtained and is digested by Bsm I and Eco RV as indicated in the figure.synthetic oligonucleotides can easily be inserted in these vectors,allowing flexibility to create all required fragment lengths.Probes consisting of two synthetic oligonucleotides and resulting in amplification products of94–124bp were also successfully used.The non-hybridising stuffer sequences of our M13-derived probe oligonucleotides provide the advantage that the amplification characteristics of a large part of each amplicon are known.Short hybridising sequences also have an advantage in mutation detection and SNP analysis as sequences close to each other can be analysed without competition between probes with overlapping target sequences.Reaction conditionsAll M13-derived MLPA probe oligonucleotides contain the complement sequence of one of the two PCR primers at their 3′end,and will thus be linearly amplified during the PCR.This PCR primer is unlabelled in order to prevent background signals.To prevent most of this PCR primer being consumed by linear amplification,low amounts of probe oligonucleotides have to be used.If10fmol of each of the40probes were to be linearly amplified,this would consume all10pmol unlabelled primer in only25PCR cycles.Despite the use of low amounts of probes,hybridisation of probe oligonucleotides to theirtarget Figure2.Outline of the MLPA reaction.sequences has to be complete in order to obtain reproducible results.We use a 16h hybridisation period in an 8µl reaction volume containing 1–4fmol of most probe oligonucleotides.Hybridisation kinetics differed slightly for each oligonucle-otide.Some probes required the presence of up to 8fmol.Once hybridisation of all probes is complete,prolonged incu-bation did not influence relative probe signal strength.Commercially available ligases,as well as several ligases cloned at MRC-Holland,were tested for use in MLPA reactions.The NAD requiring Ligase-65enzyme chosen is active at 50–65°C,but can easily be heat inactivated before the start of the amplification reaction.Ligase-65is very sensitive to probe-target mismatches next to the ligation site (Fig.3).Within the range of 20–500ng human sample DNA,MLPA results were not influenced by the amount of DNA used.Some non-specific amplification products appeared when samples containing <20ng human chromosomal DNA were analysed.Most experiments were performed using 50–100ng human sample DNA.Probe signal strengthsThe hybridising parts of our probes were designed to detect human sequences that are present in a single copy/haploid genome.However,the relative signal strength of different probes (relative peak area of each probe amplification product)is not equal.Apart from the copy number of the probe target sequence,the major factors influencing the relative signal strength of MLPA probes proved to be the amount of polymerase used in the PCR and the nature of the first nucle-otide following the labelled PCR primer.The MgCl 2concen-tration in the PCR did not have substantial effects.The KCl concentration,however,influenced the relative peak sizes of some probe amplification products.The polymerase activity during the PCR influenced the relative signal strength of a minority of the probes.Relative peak areas of 5–10%of the probes made decreased >25%when 2.5-fold lower amounts of polymerase were used,whilst the relative peak areas of 2%of the probes strongly decreased when 2–4times higher amounts of polymerase were used.These probes were replaced.The influence of the nature of the first nucleotide following the PCR primer sequence on the relative peak size was unex-pected.In our short synthetic probe oligonucleotides the PCR primer sequence is immediately followed by the variable target-specific sequence.Probes in which the PCR primer sequence was followed by an adenine had a >2-fold lower average signal strength.In all 25cases examined,replacement of this A nucleotide resulted in increased signal strength.Signal strength increased in the order A <T <G <C.Average signal strength of probes in which the unlabelled primer was first elongated with an adenine residue was also significantly lower as compared with other probes.This finding may be of use for the design of primers for traditional multiplex PCR.The vast majority of recently made MLPA probes,designed to detect human single copy DNA sequences on chromosomes 1–22,have signal strengths between 50and 150%of the average signal strength.This indicates a maximum difference in amplification efficiency during each PCR cycle of <2%from average for each probe amplificationproduct.Figure 3.Sensitivity of MLPA analysis to mismatches in the short probe oligonucleotide.MLPA reactions were performed on 100ng samples of human DNA.Amplification products were separated on a denaturing acrylamide gel (LICOR).Length (bp)as well as gene HUGO name is indicated for each probe amplification product.The probe mix contained 33complete probes.For seven other (underlined)probes,only the long M13-derived oligonucleotide was included in each test.The part of the gel shown shows the result of addition of different short probe oligonucleotides for two of these probes to the probe ne 1,Bax-specific short probe oligonucleotide,resulting in a 301bp extra amplification ne 2,as lane 1but with a mismatch (T/T)at the fourth nucleotide from the ligation ne 3,as lane 1but with a mismatch (A/C)at the 3′nucleotide of the BAX short probe ne 4,F3-specific short probe oligonucleotide,resulting in a 328bp extra amplification ne 5,as lane 4but with a mismatch (G/G)at the fourth nucleotide from the ligation ne 6,as lane 4but with a mismatch (G/G)at the 3′nucleotide of the F3short probe oligonucleotide.Probe specificityAs shown in Figure 3,omitting one of the short probe oligo-nucleotides,or replacing it by an identical oligonucleotide having a mismatch at the 3′end,prevented the appearance of probe amplification products.Sensitivity of probe signals to mismatches close to the ligation site depended on the amount of ligase used and the duration of the ligation reaction (not shown).Under our experimental conditions a mismatch at the 3′end of the short probe oligonucleotide completely prevented the appearance of probe amplification products.Mismatches at 4–6nt from the ligation site had no or only small effects (Fig.3).This sensitivity to a mismatch at the ligation site was,in some cases,used to distinguish target sequences from pseudogenes or related genes.Detection of trisomiesIn order to test the linearity of probe signal with small changes in target sequence copy number,we prepared a mix of 40probes,including four probes each for chromosomes X and Y and eight probes each for chromosomes 13,18and 21sequences.Part of the capillary electrophoresis patterns obtained on four DNA samples are shown in Figure 4.For relative quantification purposes we divided each peak area by the sum of all probe peak areas of that sample.The ratio of each individual probe relative area was then normalised tothatFigure 4.Detection of trisomies by MLPA.Samples containing 100ng DNA were analysed by MLPA using probe mix P001.Male and female control DNA was obtained from Promega.Blood-derived DNA from a triple X and a female triple 21individual were provided by the Department of Clinical Genetics,Free University of Amsterdam.Reactions were analysed by capillary electrophoresis (Beckman CEQ2000).In each case the female control DNA is shown in red.Probe mix P001contains 40probes.Only part of the resulting electropherogram is shown.Arrows indicate the positions of a 292bp amplification product of a probe specific for the TFF1gene on chromosome 21,a 319bp amplification product of a probe specific for the L1CAM gene on the X chromosome and a 337bp amplification product of a probe specific for the APP gene on chromosome 21.The other probes were specific for chromosome 3(283bp),chromosome 18(301and 346bp),chromosome 13(310bp)and chromosome 1(328bp).A complete list of genes in this probe mix can be found in Table 1.Table1.Detection of trisomies by MLPASamples containing∼100ng DNA were analysed by MLPA using probe mix P001.Male and female control DNA was obtained from Promega.Blood-derived DNA from triple21and triple X individuals as well as DNA from a triple13and a triple18cell line were provided by the Department of Clinical Genetics,Free University of Amsterdam.Reactions were analysed by capillary electrophoresis on a Beckman CEQ2000. Peak area of each probe amplification product was divided by the combined peak area of all40probes.The resulting relative peak area of each probe amplification product was divided by the relative peak area of that probe obtained on female control DNA.The presence of two copies of a probe target sequence/diploid genome should therefore result in a relative signal of1.00.The presence of three copies of a probe target sequence/diploid genome should result in a1.50relative probe signal.The exact sequence recognised by each probe of the P001probe mix can be found at the website.obtained on a control sample.In Table 1,this ratio is shown for each probe using female control,triple X,triple 13,triple 18and Down’s syndrome (trisomy 21)DNA.Results show that the relative probe signals obtained for each probe reflected the relative amount of the probe target sequences in the sample.The excellent reproducibility of relative signals obtained enabled the detection of a single extra copy of a probe target sequence per diploid genome.Highest deviations of the expected values were obtained for the triple 13DNA sample that was derived from a cell line.An increase in relative peak area of the chromosome 8-specific MYC probe (1.30),and a lower than expected relative peak area of the chromosome 13-specific RB1(1.30)and DLEU 1probes (1.25)might be due to chromosomal aberrations in some cells of our cell line.The DLEU1gene and the tumour suppressor RB1are located close (<1Mb)to each other.A probe specific for a different sequence of the MYC gene (relative signal 1.35)also indicated an increase in copy number of this oncogene in our triple 13cellline.Figure 5.Detection of BRCA1exon deletions by MLPA.Samples containing ∼100ng DNA were analysed by MLPA using probe mix P002.Female control DNA was obtained from Promega.Blood-derived DNA from individuals known to contain an exon 13or an exon 22deletion were provided by the Department of Clin-ical Genetics,Free University of Amsterdam.Reactions were analysed by capillary electrophoresis.Probe mix P002contains 34probes.Nine probes recognising non-BRCA1sequences on various chromosomes are indicated by a ‘c’.The exon recognised by the BRCA1-specific probes is indicated by a number.Probes for both alternative exons 1are indicated as 1A and 1B.Exon 4is not present in the normal BRCA1gene transcript.Two probes specific for the first and last parts of exon 11are included as this exon is very large (3.4kb).Exon deletions are apparent by an ∼50%reduction in peak area of a specific probe.The exact gene and sequence recognised by each probe of the P002probe mix can be found on the website.Detection of exon deletions in the human BRCA1,MLH1and MSH2genesThe human BRCA1gene is involved in hereditary disposition for breast cancer.In The Netherlands >30%of hereditary disposition for BRCA1-related breast cancer is due to deletions of one or more of the 24BRCA1exons (2).Deletion of one or more exons can be proved by Southern blotting provided that the deletion does not extend beyond the probe boundaries.Known deletions can be tested by PCR.We prepared MLPA probes for each BRCA1exon.Samples known from specific PCRs (2)to be heterozygote for a deletion of either exon 13or exon 22were easily identified by MLPA as they resulted in an ∼2-fold reduction of relative probe signal for these probes (Fig.5).DNA samples from 850individuals suspected of hereditary disposition for breast cancer have been tested by MLPA.Several new as well as several previously described aberrations of the human BRCA1gene were detected (F.B.L.Hogervorst,P.M.Nederlof,J.J.P.Gille, C.J.McElgunn,M.Grippeling,R.Pruntel,R.Regnerus,T.van Welsem,F.H.Menko,I.Kluijt,C.Dommering,S.Verhoef,J.Schouten,L.J.van ’t Veer and G.Pals,manuscript in preparation).Genomic deletions of parts of the human MLH1and MSH2genes are a frequent cause of hereditary non-polyposis colon cancer (HNPCC)(3).An MLPA probe mix was prepared containing probes for each of the 19MLH1exons and each of the 16MSH2exons,as well as seven probes for genes on other ing this probe mix,all six different known exon deletions tested could easily be identified by MLPA.Screening of a large number of DNA samples from HNPCCpatients is in progress.Results obtained on a sample known to contain in one chromosome a deletion of exons 1–6of the MSH2gene are shown in Figure 6.Detection of gains and losses of chromosomal regions For analysis of tumour DNA,three MLPA probe mixes have been made,each containing 41different probes specific for human single copy DNA sequences.Most target sequences were chosen in chromosomal regions often deleted or ampli-fied in various types of tumours.These probe mixes were used to analyse DNA from diffuse large B-cell lymphomas (DLBCL)as well as DNA from the SkBr3cell line.Results obtained with one of the probe mixes on a DLBCL DNA sample and the SkBr3DNA are shown in Figure 7.SkBr3is known to contain amplified ERBB2(HER2/neu)and MYC loci (16,17).Relative peak areas for two different MYC-specific MLPA probes were increased 5.3and 6.1times compared with control human DNA.Relative peak areas of three different ERBB2probes were increased 5.3,6.5and 6.0times,respectively.This compares well with the 6.6times amplification of ERBB2in SkBr3as measured by FISH (4).Exact amplification levels are difficult to determine as the number of chromosomal aberrations is large and the relative copy numbers are calculated by comparison with the total peak area of a sample.The SkBr3cell line is known to be substantially tetraploid and to contain several abnormal chromosomes (17).Apart from many other aberrations,our SkBr3DNA sample completely lacked the target sequence for a CDH1probe on chro-mosome 16q22.Major parts of chromosomes 7and 20appearedFigure 6.Detection of MSH2exon deletions by MLPA.Samples containing ∼100ng DNA were analysed by MLPA using probe mix P003.Female control DNA was obtained from Promega.Blood-derived DNA from an individual known to contain a deletion of exons 1–6of the MSH2gene was provided by the Department of Clinical Genetics,Free University of Amsterdam.Reactions were analysed by capillary electrophoresis (Beckman CEQ2000).Probe mix P003contains 42probes.Probes are present for each of the 19MLH1and each of the 16MSH2exons.Seven probes recognise other sequences on various chromosomes.Only part of the electropherogram is shown.The exon recognised by the MSH2-specific probes is indicated by a number.Control probes are indicated by a ‘c’.The remaining probes are specific for MLH1exons 1–9.Exon deletions are apparent by an ∼50%reduction in peak area of a specific probe.The exact gene and sequence recognised by each probe of the P003probe mix can be found at the website.。
gene expression signature 基因表达 -回复
gene expression signature 基因表达-回复基因表达（Gene Expression Signature）基因表达是指基因在细胞中转录成mRNA，并进一步转化为蛋白质的过程。
基因表达的变化可以直接影响个体的生理特征和疾病的发展。
在基因组学研究中，人们常常使用基因表达的模式或模式集合来描述特定疾病状态或生物学过程，这被称为基因表达签名（Gene Expression Signature）。
基因表达签名是通过高通量技术（如基因芯片或RNA测序）获得的基因表达数据，并使用统计学方法对这些数据进行分析得到的结果。
这些数据可以被用于识别特定疾病或生物学过程的生物标记物。
基因表达签名的发现可以帮助我们更好地理解疾病的发生机制，预测疾病的进展和预后，以及指导治疗策略的制定。
首先，为了获得基因表达签名，我们需要采集样本并进行基因表达测定。
常见的方法是通过基因芯片或RNA测序技术，获取样本中上千个基因的表达水平。
这些数据中包含了大量的信息，但直接分析这些数据是困难且复杂的。
因此，我们需要将数据进行预处理和归一化，以去除噪声并使不同样本之间的比较更加准确。
经过预处理后，我们可以进行差异表达分析，以发现在不同条件下表达差异显著的基因。
这些差异表达的基因可能是与特定疾病或生物学过程相关的候选基因。
接下来，我们可以使用一系列的统计学方法，如聚类分析、主成分分析或机器学习算法，对这些差异表达的基因进行分组和分类。
通过比较不同组别或类别之间的基因表达模式，我们可以鉴定出与特定疾病或生物学过程相关的基因表达签名。
这些基因表达签名可以包含一组上调或下调的基因，它们共同参与了某个特定的生物学过程或疾病发生的关键途径。
通过研究这些基因表达签名，我们可以更深入地了解疾病的发展机制，找到新的治疗靶点，并开发出更精确的诊断和预测工具。
除了在疾病研究中的应用，基因表达签名也可以帮助个体化医疗的实现。
通过分析个体的基因表达数据，我们可以预测他们对特定药物的反应，为患者提供更准确的个体化治疗方案。
gene expression signature 基因表达 -回复
gene expression signature 基因表达-回复什么是基因表达（gene expression）？基因表达指的是基因通过转录和翻译过程，将基因序列中的信息转化为蛋白质的过程。
在细胞中，基因是DNA分子的一部分，DNA分子则包含了编码蛋白质所需的遗传信息。
基因表达是生物体内复杂而精确的调控过程，对个体发育、生理功能和适应环境起着重要作用。
什么是基因表达签名（gene expression signature）？基因表达签名是指与特定生物学过程、疾病状态或临床表型相关联的一组基因的表达模式。
通过对大量样本的基因表达数据进行分析，可以识别出与特定生物学状态相关的不同基因组合。
基因表达签名的发现可以揭示基因与疾病之间的潜在关系，为研究疾病发生机制、预测个体疾病风险以及疾病分类和治疗提供重要依据。
如何得到基因表达签名？获得基因表达签名的常用方法是通过进行高通量基因表达分析。
常见的基因表达分析技术包括芯片芯片技术和RNA测序技术。
芯片技术利用具有数万个已知基因的DNA探针，通过与样本中的RNA分子的互补配对反应来检测和量化特定基因的表达水平。
而RNA测序则通过将RNA分子转换为DNA序列，再进行高通量的DNA测序，以获得基因表达的全面信息。
基因表达签名在生物学研究中的应用基因表达签名在许多生物学研究领域中都有广泛的应用。
首先，在疾病研究中，基因表达签名可以用来识别特定疾病的生物标记物，帮助研究人员进行早期诊断和疾病分类。
例如，在癌症研究中，通过分析肿瘤组织中的基因表达数据，可以发现与肿瘤发生和进展密切相关的基因表达签名，从而为癌症的早期检测和个体化治疗提供依据。
其次，基因表达签名也可以用来预测个体对药物的反应和治疗效果。
通过对大量患者样本进行基因表达分析，可以寻找与特定药物治疗效果相关的基因表达模式，进而为个体化药物治疗提供指导。
这种个体化治疗策略可以减少不必要的药物治疗，提高治疗效果，并降低药物不良反应的风险。
Relative quantification of CYP1A gene expression in whitefish
INTRODUCTION
Fish are among the organisms most sensitive to the toxicity of polycyclic aromatic hydrocarbons (PAHs), polychlorinated biphenyls (PCB’s), furans and dioxins, particularly at their early life developmental stages (Incardona et al. 2004; Prasch et al. 2003; Teraoka et al. 2003). Environmental exposures to polycyclic aromatic hydrocarbons (possibly in conjunction with PCBs, and dioxins) were related to epizootic outbreaks of liver neoplasia in feral fish from various regions of the United States (Murchelano and Wolke 1991; Myers et al. 1994).
determination of CYP1A mRNA levels by quantitative reverse transcription polymerase chain reaction (RT-PCR) has been added to biomonitoring studies (see for example Cao et al. 2000; Rees et al. 2003). A fairly new technology that emerged in the early 1990s is quantitative real-time PCR (Q-PCR). The Q-PCR reaction is monitored in real time by fluorescence either with the incorporation of the SYBR green dye that fluoresces only when it is intercalated into DNA or by a fluorescent probe that is complementary in sequence to the cDNA of interest (Higuchi et al. 1993; Larkin et al. 2003). Recently Rees and Li (2004) have developed and evaluated a real-time quantitative PCR assay for measuring the induction of liver CYP1A mRNA in species of three salmonid genera: Oncorhynchus, Salmo, and Salvelinus.
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Ct
24 22 90% 100%
Starting quantity
相对定量-Pfaffl法
/genomics
Efficiencytarget Fold induction =
deltaCt target (control-sample)
Efficiencyreference
单个内参基因校正可能会产生误差 •25%可能性高至3.0倍差异 •10%可能性高至6.4倍 •个别的样本差异表达相差20倍 利用多个内参基因校正 •http://medgen.ugent.be/~jvdesomp/gen orm/ •IQ 5/CFX96的软件可以计算
Multiplex RT-qPCR Results
Tissue #1: Tissue #2:
(From Standard curve)
Primer set #2 GOI
22 24
21 20
Efficiency:
Delta Ct: 2target
90% = 1.9
20-21 = -1
deltaCt target
100% = 2
24-22 = 2 4 0.53 =
Gene Expression基因表达
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实验设计是关键 设计引物横跨几个外显子，或外显子内含子连接片段
防止基因组DNA污染
DNA酶消解
避免假基因，防止基因组DNA的扩增
目的基因与内参基因相同设计参数
选择合适内参基因
Reference Genes内参基因
Gene Expression
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Questions?
(24-22 = 2)
Fold induction =
1.9reference
deltaCt reference
= (20-21 = -1)
7.5
方法比较
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1. Ct method: (no reference gene)
• Fold induction : 4
NM_004048 NM_003406 NM_012423 NM_004168 NM_0000194 NM_003194 NM_000190
Beta-2-microglobulin Tyrosine 3-monoxygenase activation protein, Zeta polypeptide (Phosopholipase A2) Ribosomal protein L13a Succinate dehydrogenase complex, subunit A Hypoxanthine phosphoribosyl-transferase 1 TATA box binding protein Hydroxymethyl-bilane synthase
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实验结果标明，很多常用内参基因的表达是随环境变化的，并且在根和叶片中可能不同呢。
常用内参基因相对表达, i.e. ACT2 (At3g18780, black), TUB6 (At5g12250, red), EF-1a (At5g60390, green), UBQ10 (At4g05320, cyan), and GAPDH (At1g13440, blue). B, 新的内参基因相对表达, i.e. At4g34270 (black), At1g13320 (red), At1g59830 (green), At4g33380 (cyan), and At2g28390 (blue).
Reference Genes-细胞或组织
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GAPDH：GAPDH mRNA在不同癌组织 (包括肺癌、乳腺癌、肾细胞癌等)中的 表达升高；在不同个体间、怀孕期间以 及细胞周期的不同阶段等变化很大 β-actin：当细胞向恶性转化时,βactin mRNA 的表达水平增加而假基因 的的存在也可干扰β-actin的检测。 rRNA和28S rRNA:rRNA的转录易 受到各种生物因素和 药物的影响。在 有丝分裂期间, 28S、18S rRNA 明显 减少或停止表达
Problem with the CT
Ct
24 22 90%
Starting quantity
Relative Quantification-扩增效率/ge Nhomakorabeaomics
Problem with the CT Slopes are not parallel
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根据实验要求选择合适内参基因
考虑生物样本复杂性，没有一个内参基因适合所有分析
内参基因稳定表达于不同类型的细胞和组织(如正常细胞和癌细胞)
, 而且其表达量是近似的,无显著性差别；表达水平与细胞周期以及细 胞是否活化无关;
Reference Genes-Plant植物
Reference Gene Validation-验证
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均一化处理样本量
RNA
浓度
细胞数 组织质量 CDNA浓度
reference genes CT值保持一致
IL1-b Target Gene
Actin Ref. Gene
Reference Gene Validation-验证
A,
Genome-Wide Identification and Testing of Superior Reference Genes for Transcript Normalization in Arabidopsis, Plant Physiology, September 2005, Vol. 139, pp. 5–17
deltaCt reference (control-sample)
Efficiency = 10-1/slope
(Pfaffl, 2001; Nucleic Acid Research)
相对定量-Pfaffl法
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Primer set #1Reference
18S
。在各种因素下，例如实验条件下细胞
周期的不同阶段,看家基因mRNA的表 达水平是变化的。
Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes:Genome Biology 2002, 3(7):research0034.1–0034.11
相对值(CT
)
不用内参基因均一化处理 以质量单位做为标准 准确量化初始材料
均一化表达(CT)
考虑初始样本差异 以内参基因做为标准 一个或多个在所有测试样本中恒定表达的已知参照基因
Relative Quantification -均一化表达
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Delta Ct:
GOI 22 24 24-22 = 2
Fold induction =
22 = 4
Relative Quantification -(2 - △ △ Ct)
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Tissue #1: Tissue #2:
1st Delta Delta Ct #1:
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Different reference genes
Fold Expression
Condition
geNorm
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Software Demo
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Switch to CFX96 Manager or iQ5 V2.0 software and demonstrate Gene Expression utilities and Gene Study functionality.
CT
假设
简单
100% efficiency 一个内参基因
Pfaffl法
扩增效率 一个内参基因
Vandesompele
扩增效率
法
多个内参基因均一化
复杂
Relative Quantification -△Ct
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Tissue #1: Tissue #2:
RT-qPCR
RT-qPCR
Reference Gene Validation-验证
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Cell Number ng RNA Control 1X 10e8 200ng Experimental 1 X10e8 200ng Cell Number ng RNA Control 1X 10e8 200ng Experimental 1 X10e8 200ng
IL1-b Target Gene Actin Ref. Gene
CT 20 20 CT 20 24
Housekeeping Genes-内参基因
/genomics
Symbol ACTB GAPD UBC
Accession No. M10098 M11167 NM_00101 NM_002046 M26880 18s rRNA 28s rRNA Beta actin
Gene Expression
Relative Quantification: Data Management & Analysis Settings
Hao Angela Field Application Specialist Gene Expression Division North China Bio-Rad Laboratories
/genomics
All cells plated same time from same suspension