PCR Amplification

Briefly, the tissue or cell DNA extract (4 μL) was mixed with Taq polymerase- containing REDExtract PCR reaction mix (10 μL, Sigma), primers (Invitrogen) for specific genes such as the Lac Z gene (20pmol each of forward and reverse primers) and the endogenous mouse intestinal fatty acid binding protein gene (Fabpi-200; 10 pmol each for forward and reverse primers) as an internal control in a final volume of 20 μL/PCR reaction. The PCR was performed on a PTC-100 Programmable Thermal Controller (MJ Research) set for initialisation at 93°C for 1 minute, followed by 30 cycles of denaturation at 93°C for 20 seconds, annealing and extension at 68°C for 3 minutes, and final hold at 4°C. A list of the primer sequences used is provided in the Appendix.

The PCR products (10 μL/lane) were separated on a 2% agarose gel in 0.5% TBE (44.5 mM Tris base, 44.5 mM boric acid, 1.0 mM EDTA) by electrophoresis (60V, 30mA, 2W) for approximately 2 hours, and visualised with SYBR Safe DNA gel stain (Invitrogen). Sizes of the PCR products were measured against a 100 BP ladder (New England Bio Labs).

2.5 Real-time quantitative PCR (qPCR)

To prevent RNA degradation, RNase was eliminated using nuclease-free plastics and water and by treating glassware and reagents with diethyl pyrocarbonate (DEPC, 0.1% v/v) - an alkylating agent which inhibits RNAses - overnight and autoclaving to break down and inactivate the DEPC. qPCR was used to measure changes in mRNA expression. A two-step method was used to allow analysis of multiple genes from the same sample.

2.5.1 RNA Isolation

Total RNA for qPCR was isolated using RNeasy MiniKit (Qiagen), following the manufacturer’s protocol for extraction of RNA from adherent mammalian cells. RNA from T2 cells was treated with DNase (Promega RQ1 RNase-free DNase) to eliminate DNA contamination. The absorbances of RNA at 260 nm and 280 nm were measured using a NanoDrop spectrophotometer to determine the resulting concentration and purity of isolated RNA. Before continuing to the reverse transcription step, the integrity of each RNA sample was determined by checking the presence of an A260/A280 ratio of 1.9 to 2.1 (maximum 2.3) - a lower ratio indicates the presence of contaminating proteins, A260:A230 ratios between 1.9 and 2.2, indicate the purity of the RNA. The RNA was also subjected to analysis on the Bioanalyzer (Agilent) and confirmed to have a 28S/18S ribosomal RNA ratio greater than or equal to 2, and an RNA integrity number (RIN) of 8-10 (10 being pure RNA).

2.5.2 Reverse transcription

For two step qPCR, reverse transcription (RT) of RNA into complementary DNA (cDNA) was performed using reverse transcriptase enzyme, as the PCR in the second step requires a DNA template. Avian Myeloblastosis Virus – Reverse Transcriptase (AMV-RT; Promega) was used according to the manufacturer’s instructions to reverse transcribe the RNA (800 ng of RNA was used for each set of samples) into cDNA using oligo-d(T)15 and random hexamers in a reaction volume of 20 μL. The stock reverse transcription reaction was diluted (1:20 in water) to minimise inhibitory effects of excess reverse transcriptase and RNase inhibitors on the PCR enzyme in subsequent steps.

2.5.3 qPCR amplification

A Rotorgene-6000 (Qiagen) was used for real-time PCR amplification. Specific oligonucleotide primers (Invitrogen) were added to SensiMix SYBR No-ROX (Quantace) and mixed with cDNA (2 μL). A standard curve was generated by amplifying the specific amplicon at known concentrations of 10- fold dilutions from 108 to 10 copies of cDNA/μL. The enzyme was activated with an initial incubation at 95°C for 10 minutes, followed by 40-50 amplification cycles, each cycle involving cDNA denaturation at 95°C for 15 seconds, annealing at 57°C for 20 seconds and extension at 72°C for 10 seconds. Four reference genes were tested initially: 60S ribosomal protein L13a (RLP13A), 18S ribosomal RNA (18S), β2 microglobulin (B2M), and succinate dehydrogenase complex subunit A (SDHA). Of these, 18S, B2M and SHDA were selected based on the average expression stability (M) derived using the GeNorm program (Vandesompele et al., 2002). Measurements of target genes were normalised to the geometric mean of the expression of the three selected reference genes using the GeNorm program. A list of the primer sequences used is provided in the Appendix.

2.6 Western blot

2.6.1 Sample preparation

Conditioned media were aspirated from cell cultures and centrifuged for 5 minutes at 600 x g to remove any dead cells and debris. The cell layers were washed twice in ice-cold PBS, scraped and lysed in RIPA buffer (Sigma) containing a protease inhibitor cocktail (Roche) and phosphatase inhibitors (Sigma). Total protein concentrations in cell lysates were quantified by the BCA protein assay using BSA as the standard (Pierce). Lysates were stored at -20°C.

In each experiment, equal amounts of protein (10 – 50 µg) were diluted to the same volume by addition of lysis buffer and loaded in each lane of the gels. Cell lysates were dissolved in 4x Laemmli sample buffer (0.25M Tris-HCL,

40% glycerol, 8% SDS, 0.04% bromophenol blue) for Tris-glycine gels or 4X NuPAGE sample buffer for NuPAGE gels (Invitrogen). For reduced samples, lysates were also mixed with 2-β-mercaptoethanol (BioRad; final concentration 20%) or NuPAGE reducing agent (Invitrogen). Samples were heat denatured at 95°C for 5 minutes for Tris-glycine gels and at 70°C for 10 minutes for NuPAGE gels. SeeBlue (Invitrogen) or Novex Sharp (Invitrogen; for NuPAGE gels) molecular weight standards were also loaded on each gel.

2.6.2 BCA Assay

The protein concentration of lysates was measured using the BCA assay (Pierce). Briefly, standards and samples were diluted, if required, in the lysis buffer used to lyse cells (RIPA buffer, Invitrogen) to a final volume of 10 μL, and added to a 96 well plate. Colourless BCA solutions A and B were mixed at a ratio of 50:1 to give a green working reagent (WR). WR (200 μL) was added to each well and incubated at 37°C for 30 min. The plate was cooled to room temperature and the absorbance of the wells measured at 560 nm on a plate reader (Dynex Technologies or MikroWin). The total protein concentration in each sample was calculated by comparison with the regression line of the standard curve and multiplying by the dilution factor of the samples.

2.6.3 Gel electrophoresis

Either pre-cast NuPAGE or Tris-glycine gradient gels were used, or gels were prepared from ProtoGel 30% (National Diagnostics), 4x Resolving Buffer (National Diagnostics), distilled water, 10% ammonium persulphate (Sigma) and N,N,N′,N′-Tetramethylethylenediamine (TEMED; Sigma). Various % acrylamide gels were used according to expected target protein size; lower percentage gels for higher MW proteins and higher percentage gels for lower MW proteins. Electrophoresis was performed at 125V for 90 minutes in Tris-glycine gels or 180V for 1 hour in NuPAGE gels (or longer for larger MW proteins), until the dye-front reached the bottom of the gel.