An Introduction to qRT-PCR Assays Using Sybr Green (Using SDS ...

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and can be fitted in when the machine is not in use. This charge is to cover thecost of the annual service contract for the machine.1. Plan ExperimentsBefore conducting the assay decide on the form of the experiment and howdata is to be analysed. This is an important aspect of the procedure, as it willaffect the conclusions that it is possible to draw from the results of the assay.The main considerations are:• Will expression be presented as relative expression or copies?• How many and which internal controls will be used?• Are quality controls necessary?a) Relative Expression vs Copy NumbersIn most cases biological experiments include a set of control samples, andthese may be used as a reference point for relative quantification. This ismost reliable when the comparison is being made of equivalent samples. Thatis, similar tissue/cell type with a relatively discrete biological stimulus.If the data is to be presented as a change relative to a control (eg:untreated or wildtype sample), then relative expression is an ideal approach.Setting up an assay for relative quantification is simplified as the assay revertsbe essentially being a normal PCR (albeit on a larger scale). The whole assayconsists of measuring target gene and internal control gene expression in bothcontrol and experimental samples, and the only additional samples requiredare no template controls (NTCs) to ensure that no contamination is present.Much of the early literature on qRT-PCR revolves arounddetermination of copy numbers of a transcript, also termed absolutequantification. However, an increasing number of studies have started toconsider the relevance of copy numbers to biological questions. Approachesto this problem range from the simple assumption that product will doubleevery cycle, to the use of plasmid or cDNA standard curves to deriveefficiency, or more complicated calculations of efficiency from amplificationkinetics (see ‘Data Analysis’ below).One of the main problems with copy numbers is that the preparationand maintenance of exact concentrations of nucleic acids is technicallychallenging. Despite its widespread use spectrophotometry is not an exactmethod for quantification, and furthermore, long-term storage of nucleicacids at low concentrations without any degradation is not as straightforwardas it may initially seem. Additionally, due to the exponential nature ofamplification that occurs in PCR, slight differences in amplification rates can
esult in dramatic differences in results. A 5% difference in PCR efficiency canresult in almost a twofold difference in expression after 25 cycles. If standardsamples and experimental samples do not exhibit comparable kinetics, thenequating cDNA samples with copy numbers determined from standards ismisleading. This is particularly true when comparing experimental cDNAsamples against purified nucleic acid preparations (eg: PCR product orplasmid preparations).As differences in RNA concentration, RNA quality and reversetranscription efficiency will also inevitably occur, an internal control istypically used to act as an RNA loading control (see below). As such, targetgene expression is normalised to internal control gene expression, and thisresults in a ratio of target gene to internal control, which is independent ofcopy number. For example, partial RNA degradation or poor RT efficiencywill both result in a reduced copy number, but will also result in acomparable decrease in internal control gene expression. As such, theexpression ratio of target to internal control will be the same.Are copy numbers essential within the context of your assay? In someassays, copy number may be meaningful, but in many cases it is not. Anotherway of thinking about this is does a measurement of the number of copies perug of RNA tell you anything more about the sample? For example, in thecontext of viral loading, copies per ug of RNA is obviously important, but inthe context of expression of an inflammatory marker in the brain followinginjury copies per ug of tissue is not especially meaningful – what is of interestis whether the expression of the marker changes following injury.As such, if copy numbers are really required then they may becalculated from molar concentration based upon the molecular weight of theamplicon and Avagadro’s constant. However, as well as constructing anaccurate standard curve, it must be confirmed that standards and cDNAexperimental samples amplify comparably. Finally, it should be realised thatcopy number calculations are not absolute in the qPCR context - they are ofcourse only relative to the standard used, and thus depend upon how suitablethis standard is.b) Internal Controls (Housekeeping Genes)A recurring question when dealing with quantification of geneexpression is “what is the most suitable internal control?” This questionreflects both a commendable desire to accurately control gene expressionassays, but also highlights a desire to oversimplify this difficult issue. Firstly,there is no such thing as an ideal internal control. Whilst numerous internalcontrols are available, none is ideal for every scenario, and all haveadvantages and drawbacks.Internal controls function as a loading control to ensure thatcomparable amounts of RNA are being measured in each case. However, how
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