QUANTITATIVE REAL-TIME POLYMERASE CHAIN REACTION (QRT-PCR)

2.8.1 RNA extraction

RNA was extracted from a pellet of 1x106 _{cells using a Qiagen RNeasy mini kit}

(Qiagen, Crawley, U.K.) according to the manufacturer’s instructions. Briefly, cells were resuspended in 350 μl buffer RLT (plus β-mercaptoethanol; Sigma). The sample was then homogenised using a QIAshredder spin column. One volume of 70% ethanol (Fischer Loughborough, U.K.) was added to the homogenised sample and 700 μl of this mixture added to an RNeasy mini column. The column was centrifuged for 15 seconds at 14000 rpm in a microfuge. Further DNA removal was carried out using the RNase-free DNase set. 350 μl buffer RW1 was added to the column and the column centrifuged for 15 seconds at 14000 rpm in a microfuge. The DNase I stock solution was added to buffer RDD according to manufacturer’s instructions and 80 μl of this mix was added to the column and incubated for 15 minutes at room temperature. Buffer RW1 (350 μl) was added to the column and the column was centrifuged for 15 seconds at 14000 rpm in a microfuge. Buffer RPE (500 μl) was added to the column and centrifuged for 15 seconds at 14000 rpm in a microfuge.

81 Another 500 μl RPE was applied to the column prior to centrifugation for 2 minutes at 14000 rpm in a microfuge. The RNeasy column was transferred to a 1.5 ml collection tube, RNA was eluted with the addition of 30 μl RNase-free water and was isolated by centrifugation for 1 minute at 14000 rpm in a microfuge, and stored at – 20°C.

2.8.2 RNA quantification

RNA samples were diluted 1 in 50 with RNase-free water (Invitrogen Gibco) in a total volume of 100 μl. The absorbance at 260 nm was measured at OD 260 and the RNA concentration was calculated using the following equation:

RNA concentration (μg/μl) = (OD260 x 40 x dilution factor)/1000.

2.8.3 Reverse transcription

cDNA was produced from 100 ng RNA using reverse transcription. Unless stated otherwise, all constituents were obtained from Invitrogen (Paisley, U.K.) and the procedure carried out as follows: 1 μl of both random primers (Promega) and deoxynucleotide triphosphates (dNTPs) (Bioline, London, U.K.) were added to 100 ng RNA, the volume made up to 12 μl with DNase RNase-free water (Invitrogen Gibco), and the mix heated to 65°C for 5 minutes, transferred to ice and centrifuged for 15 seconds at 14000 rpm in a microfuge. A solution comprising the following components was prepared: 1x buffer, 0.1 M DTT, RNase Out (Promega) and

82 Superscript. After centrifugation, 8 μl of this solution was added to the RNA, primer and dNTPs. The mix was incubated at 25°C for 10 minutes, 42°C for 90 minutes and 70°C for 15 minutes in a thermocycler.

2.8.4 β-actin PCR

To confirm that the reverse transcriptase reaction had worked, PCR reactions for β-actin were performed. The sequences of these primers were as follows:

Forward 5’ GTCACCAACTGGGACGACA 3'

Reverse 5’ TGGCCATCTCTTGCTCGAA 3'

The 1x reaction mix was prepared as follows: Taq buffer, primers (33 μM), dNTP's (10 mM), MgCl2 (50 mM), Taq polymerase, cDNA and DNase RNase-free water to 50

μl. The PCR cycle included an initial denaturation step (95°C for 2 minutes), followed by 38 cycles of 94°C for 20 seconds, 55°C for 30 seconds and 72°C for 60 seconds and a final incubation at 72°C for 5 minutes. Subsequently, 6 μl of a product was mixed with 2 μl 10x DNA gel loading buffer (Bioline), loaded onto a 1% agarose gel and electrophoresed in 1x Tris-borate-EDTA buffer (TBE) (see Appendix) at 60 V, for 45 minutes.

83

2.8.5 Agarose gel electrophoresis

Gel electrophoresis was used to analyse β-actin products. Powdered agarose (Bioline, London, UK) was dissolved by heating in 50 ml 1x TBE buffer supplemented with a fluorescent dye used for staining nucleic acids - ethidium bromide (0.5 μg/ml). The gel was set in the tank at RT and submerged in 1x TBE buffer containing ethidium bromide (0.5 μg/ml). Nucleic acid samples were combined with 1 μl of 6x gel loading dye (New England Biolabs, UK) and loaded into sample wells alongside the DNA molecular weight ladder (New England Biolabs, UK). Samples were subjected to electrophoresis at 80 V for 40 minutes and the gel visualised by UV illumination using bio imaging unit (Geneflow, UK).

2.8.6 Real-time PCR

2.8.6.1 Measurement of gene expression.

Reactions were performed using an ABI Prism 7700 sequence detector (Applied Biosystems) using the SensiFast SYBR Hi-Rox kit (Bioline, UK). Thermocycler conditions were 50°C for 2 minutes, 95°C for 10 minutes, followed by 44 cycles of 95°C for 15 seconds and 60°C for 1 minute. Each PCR reaction contained 900 nM gene specific 5’ and 3’ primers: VEGF (Hs_VEGFA_1_SG), GLUT1 (Hs_SLC2A1_1_SG), LDHA (Hs_LDHA_1_SG) (all from Qiagen), 1x SensiMixTM

SYBR Low-ROX MasterMix (Bioline) (containing pre-optimized dNTPs, MgCl2,

84 dH2O. Three biological replicates were used for each of the target genes, with each

individual assessed in triplicate. Results were normalised to the internal reference gene 18S rRNA. Control 18S reactions contained 50 nM 18S 5’ and 3’ primers (Sigma), SensiMix™ (Biolone) and cDNA in a total volume of 20 μl.

2.8.6.2 Q-PCR data analysis.

Q-PCR data was first analysed using ABI Prism 7000 software (Applied Biosystems) according to manufacturer’s guidelines. Briefly, cycle threshold (CT) values were determined for both 18S internal control and genes of interest in each sample by placing a threshold line over the exponential phase of the PCR cycle profiles. The average CT values were calculated from the duplicates. The 18S internal control value was then subtracted from the value for the gene of interest to give ΔCT values. This value was converted to fold change in gene expression relative to control using the equation:

Fold change = 2-ΔΔCT

and fold change was converted to percentage expression relative to control via multiplication by 100. The average and standard error of the mean of samples were calculated.

85