&& 查看话题
请教real time pcr问题，谢谢！
各位大虾们，小弟想咨询一些荧光定量的问题，如下：
1. RNA提取后，必须要测浓度吗，要根据浓度来决定加入多少ul的rna吗？
2. 假如cDNA中还有DNA污染，该怎么办？能在cDNA中加入DNAseI除去DNA吗，这回切割cDNA吗？
3. 我的模板稀释了四个梯度（5,25,125,625）选用18s做内参，结果CT值很大，都在30以上，后来调整了预变性时间，从5min增加到10min，CT值降为24左右，但是这对于内参基因来讲，仍然很大。我的目的基因CT值都在32-33以上。请问这是怎么回事？我的RNA用盒子提的，效果不错。反转录用的promega的m-mlv，用ologodt15（50uM）做引物。
4. 我有个同学做逆境，相同稀释倍数时，干旱处理时内参18S的CT在10左右，但做盐胁迫时相同18S的CT值竟然都在25左右，后来测了一下模板浓度， 都在ng左右，感觉差不多呀，听一个师姐说做不同的逆境，可能需要不同的引物，请问这是怎么回事呢？
小弟最近一直很迷茫，不敢轻易再上ABI7500去尝试了，因为材料有限，模板有限，不敢轻易得瑟模板。请各位大虾给予解答！谢谢！
1.需要测浓度的，理由：（1）不同的样品，用的RNA量一样（cDNA量也一样），定量结果更可靠。虽然内参可以校正样品量的差异，但是起始RNA量一样的话，结果更可靠，也更漂亮（理想情况是各个不同样品内参的Ct值非常接近）；（2）反转录酶能力不是无限的，说明书上都有说RNA的最大加入量，所以需要预先知道RNA浓度；（3）保证实验的可重复性。
2.DNA酶也会降解cDNA。不过可以用DNA酶处理RNA，之后灭活DNA酶（一般就是酚氯仿抽提），再做反转录。但是这样RNA的量损失很大，你样品少的话不推荐这样。当然通过引物设计，跨内含子，可以避免基因组DNA的扩增，不过引物有时候很难设计。最好的办法，还是好好提你的RNA，特别是加氯仿抽提之后取上清时，不要贪多，这样就能避免基因组DNA的污染了。再说了，你用试剂盒提的，如果还有基因组DNA污染，那就是操作不当啦。你样品宝贵，可以用其他材料，专门练练RNA提取。
（老婆逼睡觉，未完待续） 3. Ct值跟预变性时间关系不大。你的情况是模板浓度太低了。连稀释5倍Ct值都那么大，那起始的RNA量太少了。下次测了RNA浓度，然后按照反转录试剂盒允许使用的RNA最大量来做反转录。之后梯度稀释（一般10倍一个梯度）再做荧光定量PCR。这个算是预实验，要解决的问题是模板该用多少ul。
4.如果内参随处理不同而变化的话，那不是个好内参啊。好的内参基因，不管如何处理，只要模板量一样，它的Ct值就应该差不多。不知道你师姐说的用不同引物是不是指用不同的内参基因，这方面还是问问你师姐吧。可以做内参的基因很多，actin、GAPDH、tublin等等，如果18s确实有这样的变化，建议你还是查查文献，换其他内参。
一家之言，仅供参考，欢迎各位同行指正哈:tuzi26: 1.最还把RNA稀释到一个浓度，然后做反转录，尽力把所有的条件都统一到一个水平，这样做出来的结果会更可靠
2.做定量的时候确实要考虑基因组DNA污染的问题，如果你不想取出gDNA的话，那就在设计引物上下功夫，引物跨内含子的话，就可以排除gDNA的影响。或者，可以再反转录前取出基因组DNA，Takara公司有这种反转录试剂盒，在RT之前加一步gDNA去除的步骤，效果还可以，我就是用的这个试剂盒，挺好的。 Originally posted by 西瓜 at
3. Ct值跟预变性时间关系不大。你的情况是模板浓度太低了。连稀释5倍Ct值都那么大，那起始的RNA量太少了。下次测了RNA浓度，然后按照反转录试剂盒允许使用的RNA最大量来做反转录。之后梯度稀释（一般10倍一个梯度 ... 您好，我也顺便请教一下，我做的是原核生物的相对定量，内参基因不是就为了消除不同模板浓度带来的差异吗？那为什么还有调整RNA的浓度呢?我做和很多了，马上要写文章，但是没有调整其浓度到一致的水平，听你这么一说心里很忐忑！一定要调整吗？ Originally posted by
您好，我也顺便请教一下，我做的是原核生物的相对定量，内参基因不是就为了消除不同模板浓度带来的差异吗？那为什么还有调整RNA的浓度呢?我做和很多了，马上要写文章，但是没有调整其浓度到一致的水平，听你这 ... 您说得没错，内参基因就是为了消除模板量差异的，不过调整到同样的浓度，扩增曲线会更好看（内参基因的曲线几乎重合）。而且还有一个作用，那就是检验这次PCR是否可靠：模板量一样的话，理论上内参基因的Ct值应该都差不多，如果出现Ct值偏差很大的情况，那就有问题了。
我有一次摸索某样品的荧光定量PCR条件，实验室以前没人做过（注明：我是直接用的基因组DNA进行定量）。第一次做，内参基因的Ct值差异很大（用同样量的DNA），后面改进了样品DNA的提取方法，之后不同样品间内参基因的Ct值就很一致了。可能前面步骤导致了DNA的损失，或是有PCR反应的抑制剂吧。
试想假如我当时模板量用的不是一样的，那我就不能发现DNA提取方法需要改进，那么我得到的结果就很值得怀疑了。
我见过有实验室要求他们的学生，不同内参基因的Ct必须做到一致（SD<0.2还是多少），挺变态的，我师兄去他们那做实验都被折磨疯了：不断调样品浓度，但总也没法让内参Ct值的差异符合要求……一般实验室都只要求模板量用一样的即可，内参基因Ct值不要差太多，这种结果就接受了。像前面提到的实验室那样，通过调整模板浓度来使得内参一致，我觉得倒挺有造假嫌疑的。
您的情况，我觉得看您是否对自己的结果有自信了。我具的自己的例子，是一个特例：提取步骤里有能抑制酶活的成分，故Ct值差异引起了我对提取步骤的怀疑（毕竟是自己摸索，想得比较多）。如果您的一整套方法都很成熟了，那么内参出问题的可能性就很小，这样的结果是可靠的。毕竟，当初引入内参，为的就是消除模板量的差异，内参没出问题，那么模板量用的不一样也没关系。
前面那个“变态”的实验室，他们发表的结果里需要有扩增曲线的图，不得不把图做得“漂亮”些。您的情况，我估计就放上最终定量的结果就可以了吧？不是专门研究定量技术的倒是没必要把扩增曲线图和Ct值这些放上去，那么内参Ct值有点差异倒不会影响文章发表。
其实，荧光定量PCR是一个精确的定量实验，我们后面费那么大劲，为什么不在一开始就做好“定量”呢？您说是不？ Originally posted by 西瓜 at
您说得没错，内参基因就是为了消除模板量差异的，不过调整到同样的浓度，扩增曲线会更好看（内参基因的曲线几乎重合）。而且还有一个作用，那就是检验这次PCR是否可靠：模板量一样的话，理论上内参基因的Ct值应 ... 哦，我明白了，我的只要结果就可以了，谢谢您详尽的解答 谢谢您，我明白了没收获颇丰:tiger28: : Originally posted by 西瓜 at
3. Ct值跟预变性时间关系不大。你的情况是模板浓度太低了。连稀释5倍Ct值都那么大，那起始的RNA量太少了。下次测了RNA浓度，然后按照反转录试剂盒允许使用的RNA最大量来做反转录。之后梯度稀释（一般10倍一个梯度） ... 看到小木虫上好多人在问，如果内参CT值有差异，我也有此疑问，请问西瓜斑竹，内参CT值相差多大算是可接受范围，例如ACTIN GENE。 十分感谢西瓜斑竹，学到不少东西！Caister Academic Press
Julie Logan, Kirstin Edwards
and Nick Saunders Applied and Functional Genomics, Health Protection Agency,
x + 284 (plus colour plates)
January 2009
GB &#163;159 or US &#36;319
Real-time PCR (RT-PCR) technology is highly flexible and many alternative instruments and fluorescent probe systems have been developed recently. The decreased hands-on time, increased reliability and improved quantitative accuracy of RT-PCR methods have contributed to the adoption of RT-PCR for a wide range of new applications.
This essential manual presents a comprehensive guide to the most up-to-date technologies and applications as well as providing an overview of the theory of this increasingly important technique. Renowned experts in the field describe and discuss the latest PCR platforms, fluorescent chemistries, validation software, data analysis, and internal and external controls. This timely and authoritative volume also discusses a wide range of RT-PCR applications including: clinical diagnostics, biodefense, RNA expression studies, validation of array data, mutation detection, food authenticity and legislation, NASBA, molecular halotyping, and much more.
An essential book for all laboratories using PCR.
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Comprehensive coverage of the latest real time PCR technologies and applications
17 chapters
Completely up-to-date
Internationally renowned authors
Fully illustrated throughout
Explains key principles and concepts
Covers a broad range of applications
Real time PCR in food, biodefense, clinical microbiology, diagnosis of infection and much more!
Useful reference sections
"... a comprehensive overview of the RT-PCR technology, which is as up-to-date as a book can be ..." Mareike Viebahn in
"... a useful book for students ..." from J. Microbiological Methods
"provides a dual focus by aiming, in the early chapters, to provide both the theory and practicalities of this diverse and superficially simple technology, counter-balancing this in the later chapters with real-world applications, covering infectious diseases, biodefence, molecular haplotyping and food standards." from Microbiology Today
"a reference work that should be found both in university libraries and on the shelves of experienced applications specialists." from Microbiology Today
"a comprehensive guide to real-time PCR technology and its applications" from Food Science and Technology Abstracts (2009) Volume 41 Number 6
"This volume should be of utmost interest to all investigators interested and involved in using RT-PCR ... the RT-PCR protocols covered in this book will be of interest to most, if not all, investigators engaged in research that uses this important technique ... a well balanced book covering the many potential uses of real-time PCR ... valuable for all those interested in RT-PCR." from Doodys reviews (2009)
"provide the novice and the experienced user with guidance on the technology, its instrumentation, and its applications" from SciTech Book News June 2009 p. 64
"... written by international authors expert in specific technical principles and applications ... a useful compendium of basic and advanced applications for laboratory scientists. It is an ideal introductory textbook and will serve as a practical handbook in laboratories where the technology is employed." from Christopher J. McIver, Microbiology Department, Prince of Wales Hospital, New South Wales, Australia writing in Australian J. Med. Sci. ): 59-60
An Introduction to Real-Time PCR
N. A. Saunders
The development of instruments that allowed real-time monitoring of fluorescence within PCR reaction vessels was a significant advance. The technology is flexible and many alternative instruments and fluorescent probe systems have been developed and are currently available. Real-time PCR assays can be completed rapidly since no manipulations are required post-amplification. Identification of the amplification products by probe detection in real-time is highly accurate compared with size analysis on gels. Analysis of the progress of the reaction allows accurate quantification of the target sequence over a very wide dynamic range, provided suitable standards are available. Further investigation of the real-time PCR products within the original reaction mixture using probes and melting analysis can detect sequence variants including single base mutations. Since the first practical demonstration of the concept real-time PCR has found applications in many branches of biological science. Applications include gene expression analysis, the diagnosis of infectious disease and human genetic testing. Due to their fluorimetry capabilities, these real-time machines are also compatible with alternative amplification methods such as NASBA, provided a fluorescence end-point is available.
An Overview of PCR Platforms
J. M. J. Logan and K. J. Edwards
Real-time PCR continues to have a major impact across many disciplines of the biological sciences and this has been a driver to develop and improve existing instruments. From the first two commercial platforms introduced in the mid 1990s, there is now a wide choice of instruments, which continues to increase. Advances include faster thermocycling times, higher throughput, flexibility, expanded optical systems, increased multiplexing and more user-friendly software. The main features of each instrument are compared and factors important to weigh up when deciding on a platform are highlighted.
Homogeneous Fluorescent Chemistries for Real-Time PCR
M. A. Lee, D. J. Squirrell, D. L. Leslie and T. Brown
The development of fluorescent methods for a closed tube polymerase chain reaction has greatly simplified the process of nucleic acid quantification. Current approaches use fluorescent probes that interact with the amplification products during the PCR allowing kinetic measurement of product accumulation. These probe methods include generic approaches to DNA quantification such as fluorescent DNA binding dyes. There are also a number of strand-specific probes that use the phenomenon of Fluorescent Energy Transfer. In this chapter we describe these methods in detail, outline the principles of each process, and describe published examples. This text has been written to provide an impartial overview of the utility of different assays and to show how they may be used on various commercially available thermal cyclers.
Reference Gene Validation Software for Improved Normalization
J. Vandesompele, M. Kubista and M. W. Pfaffl
Real-time PCR is the method of choice for expression analysis of a limited number of genes. The measured gene expression variation between subjects is the sum of the true biological variation and several confounding factors resulting in non-specific variation. The purpose of normalization is to remove the non-biological variation as much as possible. Several normalization strategies have been proposed, but the use of one or more reference genes is currently the preferred way of normalization. While these reference genes constitute the best possible normalizers, a major problem is that these genes have no constant expression under all experimental conditions. The experimenter therefore needs to carefully assess whether a certain reference gene is stably expressed in the experimental system under study. This is not trivial and represents a circular problem. Fortunately, several algorithms and freely available software have been developed to address this problem. This chapter aims to provide an overview of the different concepts.
Data Analysis Software
M. W. Pfaffl,
J. Vandesompele and
M. Kubista
Quantitative real-time RT-PCR (qRT-PCR) is widely and increasingly used in any kind of mRNA quantification, because of its high sensitivity, good reproducibility and wide dynamic quantification range. While qRT-PCR has a tremendous potential for analytical and quantitative applications, a comprehensive understanding of its underlying principles is important. Beside the classical RT-PCR parameters, e.g. primer design, RNA quality, RT and polymerase performances, the fidelity of the quantification process is highly dependent on a valid data analysis. This review will cover all aspects of data acquisition (trueness, reproducibility, and robustness), potentials in data modification and will focus particularly on relative quantification methods. Furthermore useful bioinformatical, biostatical as well as multi-dimensional expression software tools will be presented.
Performing Real-time PCR
K.J. Edwards and J.M.J. Logan
Optimisation of the reagents used to perform PCR is critical for reliable and reproducible results. As with any PCR initial time spent on optimisation of a real-time assay will be beneficial in the long run. Specificity, sensitivity, efficiency and reproducibility are the important criteria to consider when optimising an assay and these can be affected by changes in the primer concentration, probe concentration, cycling conditions and buffer composition. An optimised real-time PCR assay will display no test-to-test variation in the crossing threshold or crossing point and only minimal variation in the amount of fluorescence. The analysis of the real-time PCR results is also an important consideration and this differs from the analysis of conventional block-based thermal cycling. Real-time PCR provides information on the cycle at which amplification occurs and on some platforms the melting temperature of the amplicon or probe can be determined.
Internal and External Controls for Reagent Validation
M. A. Lee, D. L. Leslie and D. J. Squirrell
False negatives in PCR can occur from inhibition of one or more of the reaction components by a range of factors. Therefore applications requiring a high level of confidence in the result need to be designed to control for the occurrence of false negatives. While an external, or batch, control is often used, the ideal control is one that is included in the reaction cocktail in a multiplex format. Here we discuss the application and development of molecular mimics for use as controls in real-time PCR, and explain a number of concepts and experimental considerations that will aid in the optimisation of controlled multiplexed assays.
Introduction to the Applications of Real-Time PCR
N.A. Saunders
The technique of real-time PCR has features that make its use advantageous in a wide range of applications. A number of examples, covering the main areas of application, are given in the following chapters of this book. In this introduction the important features of these applications are discussed.
Analysis of mRNA Expression by Real-Time PCR
Stephen A. Bustin and Tania Nolan
Its conceptual and practical simplicity, capacity for high throughput, and combination of high sensitivity with exacting specificity has made the fluorescence-based real-time reverse transcription polymerase chain reaction (qRT-PCR or RT-qPCR) today's method of choice for the quantification of RNA. The technology continues to evolve rapidly with the introduction of new protocols, enzymes, chemistries and instrumentation and has become the "Gold Standard" for a huge range of applications in basic research, molecular medicine, and biotechnology. Progress is increasingly associated with an increased appreciation of the limitations associated with this technology and the need for careful experimental design, application and validation.
Chapter 10
Validation of Array Data
Elisa Wurmbach
Microarray techniques allow the parallel assessment of the relative expression of thousands of transcripts in response to different experimental conditions or in different tissues. The ability to correctly identify differentially expressed genes is limited by the signal to noise ratio, the variation in the levels of gene expression, and/or the variability in the measurements due to the assay itself. Therefore, an unequivocal identification of differentially expressed transcripts requires independent confirmation. Quantitative real-time RT-PCR (qPCR) is the method of choice because of its broad range of linearity. Furthermore, it can be easily adapted to systematically study tens to hundreds of different transcripts. The cDNA microarray technique is introduced as an example, followed by comparisons to different microarray platforms and their characteristics. Data analysis of microarray experiments will show the importance of verification of results. General differences between microarray hybridisations and PCR reactions and, in particular, the performance of different platforms are described and compared. Furthermore, the effects of increasing tissue complexity on detection of differentially expressed transcripts are elucidated with specific examples.
Chapter 11
Mutation Detection by Real-Time PCR
Elaine Lyon, Rong Mao and Jeffrey Swensen
Real-time applications for mutation detection include detecting alterations associated with inherited disease, acquired alterations in oncology, and microbial or viral mutations associated with drug resistance in infectious diseases.
Probe chemistries described for these applications include hydrolysis (TaqMan&) and hybridisation probes (FRET and Molecular Beacons).
Hydrolysis probes detect mutations by allele specific hybridisation at a specific temperature, while hybridisation probes allow dynamic detection through a temperature range.
Primer chemistries are described for allele specific amplification and Scorpion primers.
Recent progress in scanning amplicons for mutations also includes high resolution melting.
The design of each of these methods is described, along with applications.
Chapter 12
Real-Time NASBA
Julie D. Fox, Catherine Moore and Diana Westmoreland
NASBA is an isothermal nucleic acid amplification method which is particularly suited to detection and quantification of genomic, ribosomal or messenger RNA. The product of NASBA is single-stranded RNA of opposite sense to the original target. First developed NASBA methods relied on liquid or gel-based probe-hybridisation for post-amplification detection of products. More recently, real-time procedures incorporating amplification and detection in a single step have been reported and applied to a wide range of RNA and some DNA targets. Thus real-time NASBA has proved to be the basis of sensitive and specific assays for detection, quantification and differentiation of RNA and DNA targets. Molecular beacons have most often been utilised in real-time NASBA whether in commercially-available kits or as published in-house developed assays. As experience in design of molecular-beacon probes increases and fluorimeters suitable for real-time NASBA become widely available this methodology will be confirmed as a suitable alternative to real-time RT-PCR (and perhaps DNA PCR).
Chapter 13
Applications in Clinical Microbiology
Andrew David Sails
The introduction of real-time PCR assays to the clinical microbiology laboratory has led to significant improvements in the diagnosis of infectious disease.
There has been an explosion of interest in this technique since its introduction and several hundred reports have been published describing applications in clinical bacteriology, parasitology and virology.
There are few areas of clinical microbiology which remain unaffected by this new method.
It has been particularly useful to detect slow growing or difficult to grow infectious agents.
However, its greatest impact is probably its use for the quantitation of target organisms in samples.
The ability to monitor the PCR reaction in real-time allows accurate quantitation of target sequence over at least six orders of magnitude.
The closed-tube format which removes the need for post-amplification manipulation of the PCR products also reduces the likelihood of amplicon carryover to subsequent reactions reducing the risk of false-positives.
As more laboratories begin to utilise these methods standardisation of assay protocols for use in diagnostic clinical microbiology is needed, plus participation in external quality control schemes is required to ensure quality of testing.
Chapter 14
Diagnosis of Invasive Fungal Infections
D.S. Perlin
The mounting prevalence of invasive fungal disease in immunocompromised patients is exacerbated by inadequate methods for pathogen detection. PCR-based amplification approaches have been developed to address this problem because conventional methods for pathogen identification lack sensitivity, specificity and speed, and some infectious organisms are difficult to culture. PCR amplification of ribosomal genes and their internal transcribed spacer regions coupled with sequence-specific detection probes are the most reliable approaches for fungal identification.
Real-time self-reporting probes capable of single nucleotide allelic discrimination have expanded PCR applications to target mechanisms of drug resistance. Clinical applications of PCR are expanding for diagnosing invasive fungal diseases in blood and respiratory specimens at an early stage to improve treatment outcomes for high risk patients.
Chapter 15
Biodefense
Christina Egan, Nick M. Cirino and Kimberlee A. Musser
With the public's reawakened concern regarding use of biological agents as weapons, the rapid detection, discrimination, and identification of pathogenic organisms and toxins has become a priority for state and federal government agencies.
High confidence, cost effective, and near real-time diagnostic methods are essential to protecting national health security whether the target is public health, agriculture, commodities, or water supply infrastructures.
While culture-based methods have been, and will likely remain, the gold standard for microbiological diagnostics, PCR-based tests offer significant advantages in sensitivity, specificity, speed and data richness that make them invaluable to diagnostic laboratories.
In this chapter, we will describe the application of real-time PCR methods in biodefense.
We will discuss the use of real-time PCR in biodefense in terms of general workflow and processing considerations, clinical diagnostic applications, environmental diagnostic applications, and multiplex screening.
Real-time PCR assays can be either quantitative (qPCR) or qualitative, depending on whether a standard curve is included with the analytical run.
Most diagnostic and biodefense applications utilise the qualitative nature of real-time PCR as
this chapter will focus on the benefits of these types of assays.
Finally, we will consider the future uses and anticipated advances in real-time PCR applications as related to biodefense.
Chapter 16
Real-Time PCR: Application to Food Authenticity and Legislation
Gordon Wiseman
Real-time PCR is now an accepted analytical tool within the food industry. Its principal role has been one of assisting the legislative authorities, major manufacturers and retailers to confirm the authenticity of foods. The most obvious role is the detection and quantification of GMOs, but real-time PCR makes a significant contribution to many other areas of the food industry, including food safety and other speciality analyses such as the detection of common wheat adulteration in pasta and the detection of allergenic species. The role of quantitative real-time PCR in determining the actual amount of these materials, which are subject to considerable regulation, is discussed together with a consideration of the uncertainty of the methods.
Chapter 17
Molecular Haplotyping by Real-time PCR
Genevieve Pont-Kingdon, Alison Millson and Elaine Lyon
Molecular real-time PCR methods can determine whether two or more mutations are on the same or different chromosomes.
This ability to haplotype without family studies is useful for research and clinical purposes and can give an advantage over genotyping.
Haplotyping by real-time PCR with hybridization probes has been demonstrated for adjacent repeats and single base alterations, with a probe that covers both sites.
However, base alterations may be separated by distances greater than a traditional hybridisation probe will cover.
We described a probe design that covers both (or all) sites, but does not include the entire sequence between the sites.
When hybridized with the template, the template is forced to form a loop.
This "loop-out" probe will dissociate from the template as a unit, therefore allowing haplotyping of base alterations separated by over 80 bp.
Examples of haplotyping by traditional probes for adjacent sequence variants, as well as examples of "loop-out" probes are presented.
(EAN: 4 Subjects: [molecular microbiology] [pcr] [molecular biology])
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