Southerns northerns and westerns

Electrophoresis of DNA or RNA molecules through an agarose or polyacrylamide gel matrix form the backbone of some key molecular biology techniques as the speed and distance that molecules migrate is in direct proportion to their size. Hence,

Figure 28.4 GATA-4 directs expression of an a-MHC-CAT reporter when co-injected into skeletal muscle, which lacks endogenous GATA-4. Neither GATA-4 nor the cardiac a-MHC gene, which contains GATA-4 binding sites in its promoter, are normally expressed in skeletal muscle. Co-injection of a promoter-reporter construct gene containing a CAT gene under control of the wild type (WT) a-MHC promoter together with a GATA-4 expression vector results in a fourfold increase in reporter activity compared to in the absence of GATA-4. Mutation of the GATA-4 binding sites (mut) abrogates activation by the transcription factor (see Molkentin and colleagues23).

Figure 28.4 GATA-4 directs expression of an a-MHC-CAT reporter when co-injected into skeletal muscle, which lacks endogenous GATA-4. Neither GATA-4 nor the cardiac a-MHC gene, which contains GATA-4 binding sites in its promoter, are normally expressed in skeletal muscle. Co-injection of a promoter-reporter construct gene containing a CAT gene under control of the wild type (WT) a-MHC promoter together with a GATA-4 expression vector results in a fourfold increase in reporter activity compared to in the absence of GATA-4. Mutation of the GATA-4 binding sites (mut) abrogates activation by the transcription factor (see Molkentin and colleagues23).

DNA or RNA fragments can be easily separated according to size.2 Denaturing and transferring DNA fragments out of gel onto a membrane of similar dimensions to the gel is known as Southern blotting, so called in deference to its inventor, Ed Southern. The blot preserves the spatial distribution of the separated fragments and fixes them permanently on the membrane. Thus, we can separate out individual molecules from a complex starting mixture such as total genomic DNA digested with restriction enzymes and look at specific DNA sequences by hybridising the resulting Southern blot with gene specific probes. Often, the high degree of conservation between the same gene from different species can be exploited

Table 28.2 Comparison of some commonly used methods for RNA detection

Method

Protocol

Advantages

Disadvantages

Northern blot

RPA (RNase protection assay)

Polymerase chain reaction (PCR) methods

Real time PCR

Gridded array filters

Microarrays, (cDNA arrays, Gene Chip)

► Quantitative

► Reusable (limited number of times)

► Hybridise to probe

Autoradiography or phosphoimaging to detect probe signal

► Hybridise radiolabelled RNA probe with RNA

► Digest un-hybridised probe with ribonuclease (RNase)

► Separate protected probe on gel

► Detect signal by autoradiography or phosphoimaging

► Make cDNA of mRNA by reverse transcription (RT)

► Amplify specific cDNA target by PCR cycling

► Visualise PCR products on gel and by Southern blot, if necessary

► Amplify specific cDNA target by PCR

► Detect product using internal fluorescent probe (e.g. TaqMan) or by fluorescent dye (e.g. SYBR green)

► DNA copies of specific genes spotted in ► Allows analysis of several hundred gridded array on filters genes simultaneously

► Probe array with labelled cDNA made by RT of ► Many commercial companies offer RNA from a particular source pre-gridded filters (e.g. cytokine array)

► Visualise hybridised spots (signal is ► Can custom make arrays proportional to abundance of RNA

corresponding to each spotted gene)

► Highly sensitive

► Quantitative

► Reliably distinguish similar sequences and splice variants

► Highly sensitive

► Requires minimal RNA input

► Highly sensitive

► Quantitative

► Product is monitored each PCR cycle (hence "real time")

► Requires minimal RNA input

► Usually involves radioactivity

► Requires large amount of RNA

► Sensitive to degradation

► Labour intensive

► Largely non-quantitative (with exception of competitive PCR methods which require labour intensive construction and prior quantification of a competitor target molecule)

► Requires expensive real time fluorescence detection hardware

► Requires large input of RNA

► Relatively insensitive

► Semiquantitative (individual results require verification)

DNA copies of genes (or oligonucleotides) are spotted onto high density grid on glass slide Hybridisation with labelled cDNA prepared by RT of source RNA. (Typically, dual hybridisation protocols with fluorescent probes are used to look for global differences between two or more RNA sources)

► Allows simultaneous analysis of thousands of genes

► Requires expensive hardware/software or commercial service costs

to use a probe to hybridise across species on a Southern blot. For example, a new gene from heart may be identified in mouse and we would wish to analyse the gene structure of its counterpart in man. As many genes belong to extensive gene families related in terms of DNA or protein sequence (homology), this can be exploited to use one gene to identify a related family member, as in the earlier cited example of using the fly gene tinman to isolate its vertebrate homologue Nkx-2.5.21

The majority of techniques for studying the expression of a gene take messenger RNA as the starting material. As with DNA analysis by Southern blotting, RNA can be studied by northern blotting (fig 28.1). In Northern blotting, RNA molecules are resolved on agarose gels and a DNA probe for the gene of interest is then used to identify expression of the gene and to gauge the size of the transcript. By including RNA samples from various human tissues on a blot it is possible to determine in which tissues in the body a particular gene is expressed. Various methods of measuring RNA are described in table 28.2.

The expression profile of proteins can be determined in a similar way by resolving a protein mixture by electrophoresis on a polyacrylamide gel matrix under denaturing conditions (to ensure proteins migrate according to their molecular mass). A modified blot procedure (western blotting) is used to transfer the proteins to a membrane, the proteins are then renatured and target protein(s) identified using specific antibodies (fig 28.1). Analysis of protein expression is an important aspect to understanding the constituents and function of the cell, and modern techniques of analysis, referred to generically as proteomics, have been reviewed elsewhere.25

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