Measurement of Gene Expression

Measurement of gene expression was carried out on human lung samples, mouse lung samples and cultured cells. Human and mouse lung samples were flash frozen in liquid nitrogen prior to RNA extraction. Cells were cultured in 6 well plates until confluent before removal by trypsinisation (A549/LA-4), scraping (J774) and then pipetting, before immediate RNA extraction.

72 2.6.1 RNA Extraction

Frozen tissue was crushed using a pestle and mortar then transferred to a clean, autoclaved 2ml eppendorf. Cells were transferred immediately to a 2 ml eppendorf before centrifugation at 13,000 x g, 5 min, 4°C to pellet the cells, and removal of the supernatant by pipetting. 1 ml of TRI Reagent was added to the samples and mixed. TRI Reagent contains phenol, and guanidine thiocyanate, which lyse the sample and dissolve DNA, RNA and protein. Samples were then immediately centrifuged at 15,000 x g for 15 minutes at 4°C.

The clear supernatant was transferred to a fresh autoclaved 2 ml tube and left to stand at room temperature for 5 minutes to ensure the complete dissociation of nucleoprotein complexes. 0.2 ml of chloroform (free from isoamyl alcohol) was subsequently added before the tubes were shaken enthusiastically for 15 seconds. The samples were then left to stand for a further 10 minutes at room temperature followed by centrifugation at 15,000 x g for 15 minutes at 4°C. Centrifugation separated the samples into 3 phases: an organic phase (red) containing proteins, interphase containing precipitated DNA, and the upper phase (aqueous, colourless) containing RNA. The RNA containing aqueous phase was transferred to an autoclaved 1.5 ml tube and 1/10 the volume of isopropanol was added then mixed.

After 5 minutes at room temperature samples were centrifuged at 12,000 x g for 10 minutes at 4°C. The supernatant was again transferred to a clean 1.5 ml tube before the remaining volume of isopropanol was added to precipitate the RNA. The RNA was pelleted by centrifugation at 12,000 x g for 10 minutes at 4°C, and the supernatant was discarded before washing the RNA with 1 ml of 75% ethanol. The samples were centrifuged for a final time at 12,000 x g for 5 minutes at 4°C, then the ethanol was removed and the RNA was re-dissolved in 50 µl of RNAse-free water.

RNA samples were quantified using a GeneQuant RNA/DNA quantifier (Amersham Pharmacia Biotech, U.K.), and the purity was assessed by A260/A280 spectrophotometry analysis. A ratio of approximately 1.8 was expected.

73 2.6.2 cDNA Synthesis by Reverse Transcription

RNA samples were diluted to 0.1 µg/µl prior to cDNA synthesis. 5µl of diluted RNA was mixed with 1x TaqMan RT Buffer, 5.5 mM MgCl2, 2 mM dNTPs mixture, 2.5 µM random hexamers, 0.4 U/µl RNase inhibitor and 1.25 U/µl Multiscribe reverse transcriptase, made up to a final volume of 50µl per sample. The mixed samples were then incubated in a Perkin Elmer 480 thermal cycler (Boston M.A., U.S.A.) at 25°C for 10 minutes, 48°C for 30 minutes and 95°C for 5 minutes (equilibration, reverse transcription, denaturation). Samples were allowed to cool, producing 10 ng/µl cDNA, which was stored at -80°C, until needed for real time PCR.

2.6.3 Real Time PCR

Real Time PCR was performed using TaqMan reagents (Applied Biosystems). 3µl of cDNA (diluted to 2.5ng/µl) was added to 12.5µl TaqMan Universal Master Mix, 1.25µl 18s (internal control) fluorescent probe, 1.25µl of the assay on demands (AoD) and 7µl of RNase-free H2O, making a total reaction volume of 25µl containing 7.5ng of cDNA template. Mixed samples were placed in a 96-well TaqMan plate and PCR was performed using a thermal cycler (Applied Biosystems). The PCR protocol of an initial 10 minute dissociation phase at 95°C followed by 40 cycles of 95°C for 15 seconds then 60°C for 60 seconds.

TaqMan assays contain forward and reverse primers for a specific gene, and a probe with a fluorescent reporter dye (FAM) at the 5’ end and a quencher dye (TAMRA) at the 3’ end. The probe is designed to anneal to a DNA sequence in between the primers on the gene of interest. When the probe is intact the quencher dye is in sufficient proximity to the reporter dye to quench the fluorescence. As the section of cDNA is amplified by successive PCR cycles, the amount of template for the probe to anneal to increases exponentially. During each cycle the exonuclease action of the polymerase enzyme (contained in the universal master mix) degrades any probe annealed to the cDNA sequence, and consequently the quencher dye is taken out of proximity of the reporter dye, allowing the latter to fluoresce.

The level of fluorescence is thus proportional to the amount of template that has been produced, and increases with successive PCR cycles.

74 18s is measured simultaneously to the gene of interest in each sample, as a ‘house-keeping’

gene. House-keeping genes are constitutively expressed genes necessary for cellular maintenance. Measuring 18s provides an internal control for the total amount of cDNA present in each sample, allowing for deviations between samples to be corrected. The 18s probe is designed in the same way as the assay probe, except it uses VIC as a reporter dye instead of FAM. All assays were validated for multiplex reactions with 18s, to ensure there was they had equivalent reaction efficiencies. For more detail on assay validation see section 3.3.3.

2.6.4 Analysis of RT-PCR Results

RT-PCR was quantified using ABI Prism 7000 software. Fluorescence of VIC and FAM was measured during the extension phase of each PCR cycle. Critical thresholds (ct) were set for VIC and FAM (representing 18s and target gene respectively) fluorescence during the exponential phase of cDNA replication. The cycle number where the level of fluorescence reaches the relative critical threshold for 18s and AoD was recorded for each sample. The relative difference between 18s and AoD ct (Δct) was then calculated. To work out the relative original amount of template for the relevant gene the following formula was used for each sample: 2^-Δct x 10^-6.