3. MATERIALS & METHODS
3.8. Quantitative Real Time Polymerase Chain Reaction: qPCR
3.7.3. Agarose gel electrophoresis
3µl of a range-appropriate loading dye (Fermentas, Canada) was added to the amplified PCR products prior to loading. 11µl of each sample was resolved alongside a 100 bp GeneRuler molecular weight marker (Fermentas, Canada) on a 1% (w/v) agarose (Roche, New Jersey, USA) gel by electrophoresis in 1x Tris/Borate/EDTA (TBE) (Appendix 1) for 90 minute at 80V. The resultant bands were visualized by staining with a 0.005% ethidium bromide in 1x TBE solution for 10 minutes followed by destaining in 1x TBE buffer for 5 minutes (Appendix 1). Images captured using the ChemiDoc XRS Imaging System (BioRad, USA) and the Image Lab imaging software (V2.0.1) (BioRad, USA) were analysed to select the most optimal primer annealing temperatures.
3.8. Quantitative Real Time Polymerase Chain Reaction (qPCR)
Schmittgen (2001). Fold changes between wildtype, transfected and treated cells were calculated for each cell type using this method and statistical analyses were carried out on these data.
3.8.2. qPCR: Experimental Design
qPCR experimental design and all subsequent procedures were subject to conformation to the requirements set out in the Minimum Information for Publication of Quantitative Real-Time PCR Experiments (MIQE) guidelines (Bustin et al., 2009). The MIQE guidelines ensure that qPCR data is obtained, evaluated and interpreted accurately in a standardized manner. Individual gene analysis experiments were carried out using three biological repeats, with three technical repeats being carried out for each biologically variable sample set. All technical repeats were run in triplicate, with each treatment in a single qPCR experiment having triplicate reactions such that n
= 3. As per conventional PCR analyses, qPCR experimental runs included no-template (NTCs) and no-Reverse Transcriptase (NRT) controls to verify that results were a true reflection of the experimental treatments on mRNA expression and not influenced by contaminating amplicons.
Detected fluorescence in all qPCR experiments was read from a baseline of 0.2 relative fluorescence units (RFU), established by the single threshold setting on the CFX manager analysis software (BioRad, USA).
3.8.3. qPCR: Statistical analyses
Mean fold changes for triplicate experimental runs were subjected to statistical analyses in the form of either a one sample t-test or a single factor Analysis of Variance (ANOVA) in order to compare the statistical significance of any differences between the means using the PASW Statistics 18 statistical analysis program.
3.8.4. Validation the use of the 2-∆∆CTmethod in analysis of gene expression
In order for the 2-∆∆CT method (Livak & Schmittgen, 2001) to be applied in qPCR data analysis, the reaction amplification efficiency of the primers amplifying the target gene of interest must be comparatively close to the amplification efficiency of the primers targeting the selected reference gene used to normalize data against. A serial dilution of the synthesised cDNA was used to assess the qPCR reaction efficiencies of both the RpS12 reference gene and the target genes (Livak and Schmittegen, 2001).
cDNA synthesised as per section 3.6 was serially diluted to obtain 5-fold template dilutions. These dilutions were subject to qPCR using both the reference and target gene primers. The average Cq
(previously CT) values for each gene was then used to generate ∆CT value for each dilution. Average
∆CTvalues were then plotted against the numeric values of the serial dilutions as data points with the
± SEM. A linear regression was generated passing through the data set, and the absolute value of the
slope (m) was evaluated. An m-value close to zero was regarded as being the standard required in order for the 2-∆∆CT method of qPCR data analysis to be applied in assessing data generated during qPCR analysis (Livak and Schmittgen, 2001).
3.8.5. qPCR: General Protocol
qPCR was performed using one of two commercially available mastermixes. SYBR-Green JumpStart® Taq ReadyMix (Sigma-Aldrich®-Aldrich, USA) was used to quantify Msx1 expression, while FIREPol® EvaGreen® qPCR Mix Plus (no ROX) (Solis BioDyne®, Estonia) was used in reactions assessing N-cadherin and Foxc1 gene expression. Each 25µℓ reaction for Msx1 incorporated the following reagents: 10.5µl nuclease-free water (Promega, USA), 12.5 µℓ SYBR-Green JumpStart® Taq ReadyMix (Sigma-Aldrich®-Aldrich, USA), 0.5 µℓ 10 µM reverse primer, 0.5 µℓ 10 µM forward primer and 1 µℓ of cDNA template. For N-cadherin and Foxc1 gene amplification, 25µℓ reactions for were set up as follows: 17.5µl nuclease-free water (Promega, USA), 5 µℓ 5x HOT FIREPol® EvaGreen® qPCR Mix Plus (no ROX) (Solis BioDyne®, Estonia), 0.5 µℓ 10 µM reverse primer, 0.5 µℓ 10 µM forward primer and 1.5 µℓ of cDNA template. In order to optimize amplification and determine the most effective annealing temperature, prepared qPCR reactions were amplified in triplicate (with three biological and three technical repeats) under temperature gradient conditions in a Mini Opticon MJ MINI™Personal Thermal Cycler (Bio-Rad, USA).
3.8.5.1. qPCR: N-cadherin Amplification Protocol
N-cadherin was initially successfully amplified when reactions using SYBR-Green JumpStart® Taq ReadyMix (Sigma-Aldrich®-Aldrich, USA) and primers spanning intro-exon boundaries (designed using Primer3) were exposed to the following cycling conditions: an initial 2 minute melting step at 95°C, followed by 39 cycles of 94°C for 15 seconds, 64°C for 1 minute, 72°C for 1 minute, and an elongation period of 30 seconds at 72°C. A melt-curve analysis to ensure product specificity was carried out on each run as follows: DNA was heated from 50°C to 95°C with a temperature transition rate of 0.5°C every 5 seconds, and a plate read at every 1 °C temperature increment.
However, these primers ceased to amplify the target sequence, and gene analyses had to be carried out using N-cadherin primers sourced from literature (Vanselow et al., 2008). These primers successfully amplified the target sequence when 5x HOT FIREPol® EvaGreen® qPCR Mix Plus (no ROX) (Solis BioDyne®, Estonia) mastermix was used under the following cycling conditions: an initial 15 minute melting step at 95°C, followed by 39 cycles of 94°C for 15 seconds, 60°C for 1 minute, 72°C for 1 minute, and an elongation period of 30 seconds at 72°C. A melt-curve analysis to ensure product specificity was carried out on each run as follows: DNA was heated from 50°C to 95°C with a
temperature transition rate of 0.5°C every 5 seconds, and a plate read at every 1 °C temperature increment (Appendix 14).
3.8.5.2. qPCR: Msx1 Amplification Protocol
Msx1 was amplified successfully when the conventional PCR protocol described by Lazzarotto-Silva et al. (2005) was modified for use on the Mini Opticon MJ MINI™ Personal Thermal Cycler (Bio- Rad, USA) for qPCR. Product was amplified by an initial cycle of 94°C for 3 minutes, 80°C for 5 minutes, followed by 39 cycles of 94°C for 1 minute, 59°C - 64°C for 1 minute and 72°C for 1 minute and a final cycle of 72°C for 10 minutes. A melt curve analysis was run as per section 3.8.5.1 above.
3.8.5.3. qPCR: Foxc1 Amplification Protocol
Foxc1 qPCR was successfully carried out after optimization trials involving the use of non-DNAse treated cDNA, the synthesis of cDNA by two methods, trials involving several different commercial qPCR mastermixes, as well as the incorporation of a range of substances in the PCR reaction to prevent the GC-rich primers from forming secondary structures and to assist the primers to anneal to the template. The protocol used in the amplification of the target sequence included an initial denaturation step of 95 °C for 15 minutes, followed by 40 cycles of 94 °C for 15 seconds, 60°C for 30 seconds, 72 °C for 1 minute and a single plate read step, followed by a 72°C extension step for 10 minutes. Melt-curve analysis was again carried out as per section 3.8.5.1 above. Curves resulting from qPCR amplification are shown in Appendix 15.
3.8.6. Agarose gel electrophoresis
If necessary, agarose gel electrophoresis of qPCR products was carried out as described in section 3.7.3 above.