For the majority of this project the cell line MDCK (strain I; Richardson et o/., 1981) was chosen as a model system to investigate factors affecting the cell surface expression of Na,K-ATPase. A further aim, was to attempt to estimate whether any changes in the expression of the enzyme could be correlated to changes in the abundance of specific Na,K-ATPase mRNA's. The background information and characteristics of this cell line have been highlighted in section l.IV..
Optimal procedures to estimate the number of sodium pump units on the surface of MDCK cells were already available and routinely performed in this laboratory (Lamb et aL, 1981). Many procedures for the extraction of RNA exist, of which the most popular is probably the method of Chirgwin et aL (1979), employing extraction in guanidinium isothiocyanate and the separation of RNA from other cellular material by CsCl density gradient ultracentrifugation. Other methods include the use of guanidinium isothiocyanate and hot phenol (Maniatis et aL, 1982), guanidinium isothiocyanate and lithium chloride (Cathala et aL, 1983), guanidine hydrochloride (Berger and Kimmel, 1987), SDS and
hot phenol (Davies et aL, 1986) and, lithium chloride and urea (Auffray and Rougeon, 1980). The method of choice for this project was a modification of the guanidinium isothiocyanate and lithium chloride procedure of Cathala et aL (1983), which is outlined in section 2.III.ii.b.. This protocol was compatible with the laboratory equipment available although its main advantage was that it was possible to process relatively large numbers of samples simultaneously (Cathala et aL, 1983). Furthermore, the method of Cathala et aL (1983) produced higher yields of both total and poly A+ RNA than the CsQ and ethanol based method of Chirgwin et aL (1979). The CsCl based method of Chirgwin et aL (1979) was also impracticable, as only a small number of cell samples could be processed at any one time.
The most commonly used method for quantifying the abundance of specific
mRNA's, involves assessing the amount of radioactive signal produced from the hybridisation of a radiolabelled DNA or RNA probe (which is complementary to the
mRNA of interest). However, the absolute abundance of specific mRNA's cannot be
determined, because the efficiencies of total RNA extraction, poly A+ mRNA isolation, and nucleic acid hybridisation are unknown. In order to compare the abundance of
specific mRNA's between different RNA samples the comparisons have to be standardised against the abundance of a parameter which is likely to be constant between
samples. The abundance of specific mRNA’s are usually standardised against the amount
of total RNA used for each sample. An example of this type of standardisation is found in Young and Lingrel (1987), who used equal amounts of total RNA per sample for the analysis of the abundance of specific Na,K-ATPase mRNA's. When experiments are performed where the level of a specific mRNA species is likely to be modulated compared to controls, the assumption is made that, the amount of total RNA per cell remains unaltered by experimental treatments. This assumption may not always be correct, as the amount of total RNA per cell can be variable (Gick et al,, 1988b). In cell culture where the number of cells are known, the amount of total RNA per cell can be estimated. However if the efficiency of total RNA extraction is low, small changes in the efficiency would result in large percentage changes in the estimate of the amount of total RNA per cell. To minimise the effect of variations in the efficiency of total RNA extraction, procedures giving consistently high percentage yields of total RNA or poly A+ mRNA are required. The premise for the experiments undertaken in this chapter was therefore to determine the percentage yield and range of variation of the total RNA extraction and poly A+ mRNA isolation procedures.
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For the general methods used in this Chapter see Chapter 2. 3.n.i. Rabbit globin mRNA radiolabelling.
Rabbit globin mRNA was radiolabelled by the enzyme poly A polymerase (as outlined in section 2.in.ii.b.5.). For the pH]-mRNA produced by poly A polymerase to be useful for the estimation of the efficiency of RNA extraction, the radiolabelled mRNA had to be separated from unincorporated nucleotides. Several separation methods were tried, including separation by Sephadex column chromatography, and denaturing agarose gel electrophoresis. Both of these techniques proved unsatisfactory, as very little pH]-
mRNA was recovered from the column chromatography and the gel electrophoresis gave a smeared band possibly due to the differing extent of radiolabelling. However, pH]-
mRNA was efficiently separated firom unincorporated nucleotides by oligo (dT) column chromatography. Unincorporated pH]-ATP does not bind to the column, but elutes with the poly A“ RNA (in the loading buffer; see figure 1).
The radiolabelling reaction was carried out as in section 2.ni.ii.b.5.. The impure [3H]-mRNA extract from the radiolabelling reaction, which was stored in ethanol, was
UV