mRNA expression

The measurement of target gene expression by measuring mRNA is an established technique in experimental and diagnostic fields and has the ability to provide quantitative data at the level of gene expression from a very small number of cells. Ultimately, measuring mRNA expression is a tool to measure the decision-making within cells under certain conditions and allow

assessment of a multitude of parameters regarding cellular differentiation and proliferation at the same time (Zamorano, Mahesh and Brann, 1996). Within the context of this study, I am more interested in the level of functional protein produced in the context of transient manipulation of gene expression. However, mRNA expression adds valuable information to support these hypotheses as well as allowing a more detailed investigation of the more subtle interaction between the genes of interest and down-stream pathways. Clearly, taken on it is own, the quantification of mRNA expression following transient transfection does not establish a direct link between genes of interest and cellular behaviours as multiple steps following gene transcription will dictate whether functional proteins will be produced. mRNA expression data does have the potential, however, to add weight to some of the theories regarding the interactions between genes and to the extent that these interactions are ligand driven (Bustin, 2000).

Reverse transcription polymerase chain reaction (RT-PCR) can involve either a one or two step technique and when combined with Taqman chemistry, can give reliable real time information regarding mRNA expression. The fundamentals of the procedure involve the creation of a cDNA library from RNA extracted under various experimental conditions prior to conventional PCR in combination with a labelled probe. In this case, specifically designed Taqman probes measuring (Applied Biosystems, Paisley, UK[9]). Reverse

transcription was catalysed using avian myeloblastic virus reverse transcriptase (AVM-RT: Applied Biosystems; Paisley, UK) which has higher fidelity,reducing issues with RNA secondary structure than other reverse

transcriptases (Freeman, Vrana and Vrana, 1996). As cDNA libraries are much more stable they can be stored at -20 ̊C and revisited as needed.

The Taqman assays for ERα, ERβ and RET were obtained and used as per manufacturers guidelines (Invitrogen; Paisley, UK). The assays consist of a fluorescence labelled dye and a quencher on either end of probes designed specifically to the ‘5 end of the amplicon (Holland et al., 1991). Primers are designed for either end of the amplicon on separate exons. During thermal cycling, taq polymerase reconstitutes the amplicon. As the fluorescent probes have a lower affinity than polymerisation, the probe is displaced and hydrolysed, first at the fluorescence end and then at the quencher end of the probe (Bustin, 2000). As the dye and quencher are separated the fluorescence intensity can be measured and is directly proportional to the number of molecules that bound probes during the cycle (Bustin, 2000). Fluorescence intensity machinery is specifically designed to perform thermal cycling as well as fluorescence detection and quantification. As the PCR amplification is very sensitive at amplifying small quantities, contamination remains a key issue in setting up experiments. The single analyser and cycler set up, allowing reactions to take place in a single sealed well of a 96 well plate without the need to potentially introduce contamination goes some way to ameliorating this. (Bustin, 2000).

7.1.5 Immunohistochemistry

It is important within the context of a clinical based study such as this to make an attempt to tie back the findings in the laboratory with the clinical

parameters and observation with blood and tissue samples collected so far. Immunohistochemistry is a technique that, used in isolation, can have considerable issues with regard to establishing scientific basis of diseases (Tavangar et al., 2007). It is, however, a useful technique in some situations and can provide valuable corroborative evidence regarding causative links. The major limitation to the use of immunohistochemistry within the context of this study is the reliability (sensitivity and specificity) of the staining techniques in verifying true positives and negatives particularly in the context of historical blocks and slides. Particular issues arise when using staining techniques and looking for protein expression beyond that which is used in the diagnostic setting and in addition, using antibodies that are not optimised for a specific tissue .

With respect to the tumour library available for this project, the most interesting results would be to look at the ERβ characteristics between family members with a known ERβ mutation and the wider cohort of patients with MTC, as well as looking specifically at RET expression levels in relation to this. Neither RET nor ERβ staining is optimised for use within the clinical setting and as such, challenges exist in extraction of meaningful and comparable results.

7.2 Methods

7.2.1 Cell culture

Cells were maintained and passaged as described in Chapter 2.

7.2.2. Site directed mutagenesis

Primers were designed as previously described to induce the two ESR2 mutations identified within the cohort. PrimerX software was used to model the identified mutations (web based SDM primer designing tool; bioinformatics.org, USA) and optimised using for melting temperature and GC content to improve binding capability using the following equations.

Tm = 81.5 + 0.41(%GC) – (675/N)

Tm = 81.5 + 0.41(%GC) – (675/N) - %mismatch

Primers were purified using polyacrylamide electrophoresis (PAGE) to improve fidelity prior to use.

Table 32. Site directed mutagenesis primer design

Primer Name Sequence Melting Temp °C % GC content Flankin g region Mutatio n Purification type 948delT forward GGCCAAGAAGAT CCCGGCTTTGTG G 77.8 60.0 12bp Single base deletion PAGE 948delT reverse CCACAAAGCCGG GATCTTCTTGGC C 77.8 60.0 12bp Single base deletion PAGE V128L forward GACACTGAAAAG GAAGCTTAGTGG GAACCGTTG 77.7 48.5 16bp Single base substitut ion PAGE V128L reverse CAACGGTTCCCA CTAAGCTTCCTTT TCAGTGTC 77.7 48.5 16bp Single base substitut ion PAGE

7.3 Results

7.3.1 MCF 7 cells were the most reliable cell type for ER receptor