EL ESCENARIO URBANO.

RT-PCR is used as a method for the quantification of mRNA control. A critical stage in relative quantification design is the selection of an appropriate internal standard, sometimes known as endogenous controls (Thellin et al., 1999). All relative RT-PCR require internal standards, mainly housekeeping genes, so called because their synthesis occurs in all nucleated cell types since they are necessary for the cell survival. The synthesis of those molecules is often considered as being less erratic in comparison to that of others and, by their commonplace use, their expressions are considered as constant and secure. However numerous studies have indicated that even in these genes, their expression does vary in given situations between different tissues and between the same tissue under different conditions (Huitorel and Pantaloni, 1985, Chang et al., 1998, Hobbs et al., 1993).

RT-PCR-specific errors in the quantification of mRNA transcripts are easily compounded by any variation in the amount of starting material between samples. This is especially relevant when the samples have been obtained from different individuals, and will result in the misinterpretation of the expression profiles of the target genes. Therefore endogenous control expression levels must be the same in all samples in the study. It is therefore important to determine if the study treatment is affecting the expression level of the candidate endogenous genes. Consequently, an important aspect of experimental design in this thesis is determining an appropriate internal standard for chondrocyte studies, that is expressed at a constant level and which is unaffected by the changes in osmolarity.

In previous studies in chondrocytes looking at gene expression in osmolarity experiments GAPDH has been used (Aigner and Dudhia, 2003, Tew et al., 2007, Hung et al., 2003, Palmer et al., 2001). Therefore, prior to commencement of gene expression studies the validation of GAPDH as an appropriate internal control was investigated.

A human endogenous control plate, part number 4396929 (Applied Biosystems, Warrington, UK) was used to undertake this study. The plate evaluates the

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expression of thirty two select housekeeping genes in total RNA samples using RT- PCR.

The following genes were evaluated;

ACTB POP4 PSMC4 PPIA IP08 RPS17 GUSB TBP

PGK1 GAPDH ELF1 CDKN1B TFRC PUM1 MRPL19 CDKN1A

UBC B2M RPL37A EIF2B1 POLR2A YWHAZ PES1 ABL1

CASC3 RPLP0 HPRT1 MTATP6 GADD45A HMBS RPL30 18S

Samples of cDNA from human articular chondrocytes exposed to 380mOsm and 550mOsm with and without the MEK/ERK inhibitor U0126 were used in duplicate. Total RNA was prepared from monolayers in culture plates using Tri Reagent (Ambion, Warrington, UK). The Guanidium-thiocyanate-phenol-choloform extraction technique was used as previously described (Chomczynski and Sacchi, 1987). M-MLV reverse transcriptase and random hexamer oligonucleotides were used to synthesize cDNA from RNA (both from Promega, Southampton, UK) .Aliquots were amplified by PCR in 20μl reaction volumes on an ABI 7700 Sequence Detector using a Taqman gene expression mastermix (Applied Biosytems, Warrington, UK).

Data was analyzed by first determining the average CT of each sample for each control gene. A ratio was then made by successively dividing each sample by the first for each control gene. This data was then manipulated in order to run it through the GeNorm programme (www.medgen.ugent.be/~jvdesomp/genorm/). GeNorm is a collection of ‘Visual Basis for Applications’ (VBA) macros for Microsoft Excel to determine the most stable reference internal control genes from a set of tested candidate reference genes in a given sample panel. From this, a gene expression normalization factor can be calculated for each sample based on the geometric mean of a user-defined number of reference genes. The underlying principles and formulas have been described (Vandesompele et al., 2002).

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Figure 1: Average expression stability values (M) of the candidate reference genes. Average expression stability measure (M) of control genes during stepwise exclusion of least stable reference genes. M represents from the least stable (left) to the most stable (right), analysed by the geNorm programme.

The results are expressed as ‘Average Expression Stability’ (M). Within this system the least stable gene has the highest M value and the most stable gene has the lowest M value. To assess the validity of the established gene-stability measure, that is, that genes with the lowest M values have the most stable expression, the gene specific variation for each control gene is determined as the variation coefficient of the expression levels after normalization. This coefficient should be minimal for proper housekeeping genes. It has been proposed that M values inside the ranges M ≤ 1, are acceptable (Hellemans et al., 2007). Interestingly 18S which has been used in a wide variety of studies in various tissues (Gorzelniak et al., 2001, Liu et al., 2005) had a relatively high value of 0.06 and expressed the 5th worst gene stability. The findings

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confirmed that with an M value of 0.035, GAPDH was a suitable normalisation factor for the osmolarity studies in chondrocytes. The majority of studies quantifying gene expression in connective tissues also useGAPDH as the reference gene (Ayers et al., 2007).

Despite many qRT-PCR studies having reported the use of a single endogenous control gene (Suzuki et al., 2000), a normalisation strategy based on a single housekeeping gene may lead to erogenous errors (Vandesompele et al., 2002, Tricarico et al., 2002). Hence the use of a panel of reference genes has been proposed (Vandesompele et al., 2002, Thellin et al., 1999). The geometric mean of multiple carefully selected housekeeping genes would then be calculated. However as many gene expression studies in cartilage osmolarity studies have used GAPDH as a single reference gene (Tew et al., 2009, Hung et al., 2003, Palmer et al., 2001) and following the validation using the endogenous control plate of GAPDH in this thesis GAPDH was used as a single endogenous control gene for this study.

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Appendix 2