Laboratory Techniques

2.3.1 Quantitative polymerase chain reaction (qPCR)

RT-qPCR is a technique facilitating quantitative mRNA expression analysis through combining PCR amplification and detection.

32 2.3.1.1 Primer design and validation

FASTA gene sequences from NCBI (http://ncbi.nlm.nih.gov) for mouse and rat were inputted into NCBI Primerblast (http://www.ncbi.nlm.nih.gov/tools/primer-blast/).

Primers were designed to meet the following criteria: be approximately 20 base-pairs (bp) long, resulting product ~200 bp, low self-complementation and no potential hairpin generation. Two primer pairs were selected per gene and then tested for

homology elsewhere in the genome via BLAST

(https://blast.ncbi.nlm.nih.gov/Blast.cgi). Primers were then commericially synthesised by Sigma-Aldrich (Dorset, UK). Primer stocks were stored at -20°C and working solution aliquots (10 µm) were used to minimise freeze-thaw cycles. Primer validation was then conducted to assess amplification efficiency and simple amplicon specificity (detailed 2.3.1.3).

2.3.1.2 RNA isolation, DNase treatment and cDNA synthesis

For both species flash frozen PFC, hippocampus and cerebellum samples from dissected brains were removed from the -80 °C and kept on dry ice. ≤30 mg sample was processed using Qiagen’s RNeasy kit. 14.3 M ß-Mercaptoethanol was added to the supplied RLT buffer (1:100) and 550 µl of resulting solution added to the sample in a ribotube (MP Biomedicals, UK). The ribotube containing the sample was placed in a ribolyser (Bio-Rad Laboratories Inc., USA) and tissue homogenised using the fast prep homogeniser for 2 x 5 second blasts until the tissue was completely lysed.

Samples were then centrifuged for 3 minutes at 14,000rpm for 3 minutes. The solution was then transferred to a 1.5ml Eppendorf, before then centrifuged again for 3 minutes at 14,000rpm to form the pellet. The resulting supernatant was dissolved in 500µl 70% ethanol and 500µl of the supernatant/ethanol mix and loaded into a RNeasy spin column in a collection tube. This column was then centrifuged for 15 seconds at 10,000rpm. The flow through was discarded and this step repeated with the remaining 500µl supernatant. 250µl of buffer RLT + ß-Mercaptoethanol was then added to the pellet and centrifuged for three minutes at 10,000rpm. The supernatant was once again extracted, mixed with 250µl 70% ethanol and added to the same RNeasy column, and again centrifuged for 15 seconds at 10,000 rpm and the flow through discarded. The column was then incubated at RT for 5 minutes. Following this the column was washed by adding 700µl Buffer RW1 to the column and centrifuging for 15 seconds at 10,000rpm and discarding flow through. 500µl Buffer RPE was then added, centrifuged for 15 seconds at 10,000rpm and flow through discarded. Another 500µl RPE was added to the spin column and the column

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centrifuged for five minutes at 10,000rpm to thoroughly wash the membrane. The column was then placed in a new collection tube and span at 14,000rpm for 1 minute to eliminate any possible carryover of Buffer RPE. The spin column was then placed into a 1.5ml collection tube. 30µl of RNase-free water (Ambion Life Technologies, UK) was added directly to the column membrane and incubated for 10 minutes at RT. To elute the RNA, the column was centrifuged at 10,000rpm for 1 minute. RNA content was then measured on a NanoDrop spectrophotometer (Thermo Fisher Scientific, Delaware, USA) to determine concentrations were a suitable level (between 100-10,000ng/ul). RNA purity was then determined by confirmation that the A260/A280 was below 2. A minimum concentration of 75 ng/µl was required for cDNA synthesis.

To remove any contaminating DNA the RNA samples were DNase treated with an Ambion TURBO DNA-free™ Kit (ThermoFisher Scientific, Delaware, USA). 5 µl 10x TURBO DNAse buffer and 1 µl TURBO DNAse were added to each sample and mixed. Samples were then incubated at 37°C for 30 minutes. 5.5 µl DNAse TURBO Inactivation Reagent was then added to each sample and mixed. Samples were incubated at RT for 5 minutes then centrifuged at 13, 000 rpm for one minute. The supernatant was then transferred to a new tube.

The eluted RNA was then used to generate cDNA. To achieve a reaction volume of 20 μl the volume of eluted RNA required for 1.5 ng/µl was calculated and added to random primer cDNA synthesis tubes (Takara Clontech, France) with RNAse free water (Ambion Life Technologies, UK) making up the volume. Samples were then placed in a thermal cycler (Bio-Rad Laboratories S100, USA) on an optimised cDNA synthesis program: 42°C for 75 mins, 80°C for 15 minutes and infinite sample holding at 8°C. 20 µl cDNA was added to 480 µl qPCR grade water for a 1:15 dilution.

Samples were stored at -20°C.

2.3.1.3 Standard Curves

A 1:5 serial dilution series across 6 points of known concentration template cDNA for each species in each brain region investigated (PFC, hippocampus, cerebellum) was used to generate a standard curve for each primer pair for each gene. Each run included measuring the expression of two validated house-keeping genes (Hypoxanthine-guanine phosphoriobosyltransferase – Hprt, Glyceraldehyde 3-phosphate dehydrogenase – Gapdh, Polyubiquitin-C - Ubc) (See Table 2 for sequences). The primer sequences of the genes of interest (Dlg1 – Dlg4) are detailed in the appropriate Chapters (mouse – Chapter 3 3, rat –Chapter 6).

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Gene Species Forward Reverse

Gapdh Rat TCTCTGCTCCTCCCTGTT

Table 2. Primer sequences for validated qPCR probes in both species.

To generate standard curves each well of a 96 well plate contained 15 μl reaction mixture (1.9 μl sterile RNase free water, 0.3 μl 10 μM forward primer, 0.3 μl 10 μM reverse primer, 7.5 μl SensiMix (Bioline) and 5 μl cDNA or water (for no template control). After loading, plates were centrifuged at 3,000 rpm for approximately 10-20 seconds before being transferred to Real-Time PCR instrument (Applied Biosystems) on a standard run: 95°C for 10 minutes, followed by 45 cycles of 95°C (15 seconds) and 60°C (1 minute) to allow for duplex denaturing and annealing and elongation, respectively. Finally a melt curve was obtained. Samples were heated to 55°C for one minute and 95°C for 15 seconds. This final stage allowed assessment of single amplicon specificity – when the resulting dissociation curves were visualised as a single peak the primers are considered specific to the target cDNA. The Ct values measured were plotted against initial input amounts on a semi-log10 plot, fitted to a straight line and a gradient generated. This gradient was then inputted into the

Thermoscientific efficiency calculator.

(https://www.thermofisher.com/us/en/home/brands/thermo-scientific/molecular-

biology/molecular-biology-learning-center/molecular-biology-resource-library/thermo-scientific-web-tools/qpcr-efficiency-calculator.html). Primers with efficiencies between 90-110% were considered valid and any primers with abnormal melt curves, low efficiency or vastly differing efficiency across regions were redesigned.

2.3.1.4 RT-qPCR Experiments

Each well of a 96-well plate contained 15μl reaction mixture (1.9 μl sterile RNAase free water, 0.3 μl 10 μM forward primer, 0.3 μl 10 μM reverse primer, 7.5 μl SensiMix (Bioline, London, UK)) and 5 μl cDNA or water). Only primer pairs previously validated

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were used. The gene of interest values were normalised to the housekeeping gene controls (Gapdh Ubc). After loading plates were centrifuged at 3,000 rpm for approximately 10-20 seconds before being transferred to the q-PCR machine.

2.3.1.5 RT-qPCR Analysis

The cycle threshold (Ct), the value reflects the number of cycles it took for the detection of cDNA signal above the background fluorescence, is outputted by the qPCR machine. There is a negative correlation between the amount of cDNA in the sample and the Ct values. The threshold levels were set at the beginning of the exponential phase and conserved across plates. Quantification was conducted using the comparative Ct method (2^-ΔΔCt method) to produce fold changes in control and Dlg2 heterozygotes (Schmittgen and Livak, 2008). The 2^-ΔΔCt method involves subtracting the average housekeeping gene from the gene of interest (Δ Ct = Ct target

– Ct reference). The geometric mean of two housekeeping genes was used throughout this thesis. ΔΔ Ct is then calculated by subtracting the experimental group from the control group (ΔΔ Ct = ΔCt test – ΔCt control) and incorporating standard deviations into the fold change.

2.3.2 Immunohistochemistry

Perfused brains were embedded in OCT and frozen. Prior to sectioning they were removed from the -80°C and placed in the cryostat (Leica Microsystems CM1860UV) for 20-30 minutes to warm to temperature (normally between -20°C to -25°C). All brains were coronally sectioned at 40 µm. Co-ordinates for the start and end of the brains regions of interest are detailed in appropriate Chapters (4 and 5). Free floating sections were placed into 1x PBS in 12 well plastic plates and stored at 4°C.

2.3.2.2 Immunohistochemical staining

Sections were blocked in 500 μl phosphate buffered saline with 1% Tween 20 (PBST) containing 3% normal donkey serum (S30-100ML, Millipore, Hertfordshire, UK) at RT with agitation for 2 hours. Primary antibodies (described in Chapter 4 and 5 methods) were diluted in 500 μl 0.1% PBST with 0.2% normal donkey serum (v/v) and incubated overnight with agitation at 4°C. Sections were washed 3 times for 10 minutes in 1x PBS. Alexa Fluor® secondary antibodies (ThermoFisher Scientific,UK) were diluted (1:1000) in 500 μl 0.1% PBST with 0.2% normal donkey serum. Sections were protected from light and incubated at RT with agitation for 2 hours. Sections were incubated with the nucleus DNA stain 4′,6-diamidino-2-phenylindole (DAPI) (1:1000,

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D9542-10MG, Sigma-Aldrich, Dorset, UK) in 500 μl 1x PBS at RT with agitation for 5 minutes, then washed 2 x 10 minute in 1 x PBS. Sections were mounted in a counterbalanced manner with 20 μl Mowiol® (4-88, Sigma-Aldrich, Dorset, UK) added per slide and glass cover-slipped and stored at 4°C. Image acquisition, and data sampling and analysis for each experiment is detailed in Chapters 4 and 5.