DEMONSTRATION BOOKLET
In situ Hybridisation
In situ hybridisation allows the visualisation of gene expression in sectioned or whole-mounted material, and thus permits the identification of the cell types in which a gene is expressed at any particular stage. Specimens (whole or sectioned embryos, brain slices, sectioned organs from adults, etc) are hybridised at an appropriate stringency with labelled RNA probes. Originally, probes were labelled with radioactive nucleotides, but more normally these days they are labelled with chemically modified nucleotides, and these are then detected using an antibody specific to the modification.
8) Consider Fig. 4, expression of WT1 mRNA in the developing human kidney. Panels A and B show low power views of sections through the whole kidney, whilst C-H show high power views of three stages in glomerular development. By reference to the text of the original paper (Mundlos et al, 1993, Development 119, 1329-1341), state in which cell types WT1 mRNA is expressed. (2 marks)
Antibody staining
Antibodies specific to particular proteins can be used to stain whole embryos or sections of tissue to examine where the protein is expressed. Fixed embryos or sectioned material is incubated with the antibody under conditions where the specific antigen are recognised, excess unbound antibody is washed off, and bound antibodies are detected using enzymatic labels.
9) Consider Fig. 5, WT1 protein expression in the developing human kidney. Is the expression of WT1 protein in the three stages of glomerular development consistent with the expression pattern of WT1 mRNA shown in Fig. 4? (1 mark)
10) Consider Fig. 6, WT1 protein expression in the adult and fetal podocytes. The figure shows that WT1 protein is localised to the nucleus of these cells. What is one advantage of antibody staining over in situ hybridisation? Is this consistent with what you know of the WT1 gene sequence? (3 marks)
Nuclease protection
S1 nuclease protection is used to define the positions of 5’ ends and intron boundaries of RNA relative to specific sites, such as the positions of restriction enzyme cleavage sites. By using RNA from specific tissues or from embryos of different ages, mRNA variants that show tissue-specific or temporal variations can be analysed. A radiolabelled single-stranded DNA probe corresponding to the region of interest (usually a restriction enzyme fragment flanking the expected transcription start site or intron-exon boundary) is made and hybridised to total RNA extracted from the stage or tissue of interest. The resultant DNA-RNA hybrid consists of a double-helical core with single stranded tails on each end. The single-stranded regions are then cleaved with S1 nuclease, and the resulting protected fragment is run on a denaturing polyacrylamide gel, which is then dried and exposed to X-ray film. Using suitable size markers, the size of the protected radioactive fragment can be readily estimated, and thus positioned with respect to the restriction enzyme sites used. A modification of the method produces a very sensitive quantitative technique for accurately measuring RNA levels.
11) Consider Fig. 7, S1 analysis of the 5’ end of the WT1 gene in testis, ovary and liver. Firstly, examine the control lane (-S1) in B. This represents the undigested probe. A) What size was the probe? B) What restriction enzyme fragment does it represent? (2 marks)
12) Describe the distribution and relative abundance of each of the two initiation variants, labelled I and II. (2 marks)
Comparing different methods
These different methods of analysing gene expression in some cases appear to give the same information. But are they are redundant? What are the advantages and disadvantages of each method?
13) Fill in the following table with as many (at least three) advantages and disadvantages as you can think of. Feel free to discuss your ideas with the demonstrators. If you require extra space to complete this question then you can attach additional sheets of paper to the booklet. (20 marks)
Method
Advantages
Disadvantages
Northern analysis
RT-PCR
In situ hybridisation
Antibody staining
S1 nuclease protection
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