A small point was painted on their upper face (Figure 32) with a non-toxic marker. Eggs were always maintained with
3.3.5. Relative quantification and statistical analysis
Relative quantification is based on determination of a target gene expression (MC1R) depending on the expression of this gene compared to a control sample or calibrator. In addition, a housekeeping gene (ACTB) is necessary as internal control and to normalise the expression of the target gene (Pfaffl, 2006). In this study, the objective was to analyse MC1R gene expression in melanic and non-melanic populations to assess whether there are different levels of expression in both adult individuals (tails) and in different development stages (eggs).
In the analysis of adult individuals based on tail samples, the calibrator sample was D10, a non-melanic form from Dragonera Island; and in the analysis involving different stages of embryonic development (eggs), the calibrator sample was from Colom Island (Colom11_1), also a non-melanic population. In
67
Materials and Methods RT-qPCR a positive reaction is detected by accumulation of fluorescent signal. Ct values (cycle threshold) (Pfaffl et al., 2004) is the number of cycles at which the fluorescence emitted was higher than the fluorescence detection threshold (exceeds background level). Ct levels are inversely proportional to the amount of target gene in the sample, so lower Ct values indicate higher levels of gene expression. For each sample, the average Ct was calculated between replicates of MC1R and normalised using the average Ct value of the reference gene, ACTB. Relative quantification was conducted using two approaches:
The standard 2-(∆∆CT) method (Livak & Schmittgen, 2001),
∆∆CT = (Ct
target– Ct
reference)
Time X– (Ct
target– Ct
reference)
Time 0Average Ct values of melanic or non-melanic samples (Time X) were normalized using reference gene (ACTB) and depending on the calibrator sample (Time 0). The results were presented as fold change (2-(∆∆CT), i.e. as the expression ratio. Positive values of fold change indicated an upregulation of the target gene, while a negative value represents a low expression of that gene (Livak & Schmittgen, 2001). Some factors can bias the fold change of the analysis, such as the PCR efficiency or the absence of expression.
The other method used was Pfaffl method (Pfaffl, 2001), where PCR efficiencies of target and reference gene were taking into account to calculated the ratio of relative expression,
PCR efficiencies (E) were calculated by performing a standard curve using six 1:10 dilution series run in triplicate for each gene (MC1R and ACTB). PCR efficiency is affected by different factors, like the specific use of PCR machine, reagents, or primers. A tail sample from a non-melanic P. lilfordi population from Rei island (Menorca) was used to perform the standard curve for the gene expression experiment based on tail samples. In the analyses based on egg samples, the standard curve was carried out with a sample from the melanic population of Aire (Aire25_2) in Menorca (Figure 36).
In order to determine whether the different values found in expression levels between melanic and non-melanic groups were statistically significant or were the result achieved by chance, the p-value of each group compared in the analysis was calculated with a statistical test. A p-value lower than 0.05 is considered significant, according to the gold standard in statistics (Fisher, 1925). The statistical test used can be parametric or non-parametric. Before choosing the test to use, we needed to determine whether the distribution of the Ct values for every comparison followed a “normal” distribution. The parametric test runs under the assumption that the distribution is normal while non-parametric tests do not make such assumption. When normality is not proven, using a non-parametric test (not assuming normality) reduces the risk of misinterpretation of the results.
A Kolmogorov-Smirnov or a Shapiro Wilk’s test with a p < 0.05 was carried out to verify whether fold change values in expression levels in melanic and non-melanic groups followed a normal distribution.
If the results did not follow a normal distribution, the Mann-Whitney test was performed to compare medians between melanic and non-melanic groups. One-way ANOVA and t-test were used as a parametric method when the data seemed to follow a normal distribution. All statistical test were carried out in R (R Core Team, 2018).
characteristic and depends on GC content or PCR product length. Each sample was analysed in triplicate and no-RT and no-template controls were included in all experiments.
3.3.5. Relative quantification and statistical analysis
Relative quantification is based on determination of a target gene expression (MC1R) depending on the expression of this gene compared to a control sample or calibrator. In addition, a housekeeping gene (ACTB) is necessary as internal control and to normalise the expression of the target gene (Pfaffl, 2006). In this study, the objective was to analyse MC1R gene expression in melanic and non-melanic populations to assess whether there are different levels of expression in both adult individuals (tails) and in different development stages (eggs).
In the analysis of adult individuals based on tail samples, the calibrator sample was D10, a non-melanic form from Dragonera Island; and in the analysis involving different stages of embryonic development (eggs), the calibrator sample was from Colom Island (Colom11_1), also a non-melanic population. In RT-qPCR a positive reaction is detected by accumulation of fluorescent signal. Ct values (cycle threshold) (Pfaffl et al., 2004) is the number of cycles at which the fluorescence emitted was higher than the fluorescence detection threshold (exceeds background level). Ct levels are inversely proportional to the amount of target gene in the sample, so lower Ct values indicate higher levels of gene expression. For each sample, the average Ct was calculated between replicates of MC1R and normalised using the average Ct value of the reference gene, ACTB. Relative quantification was conducted using two approaches:
The standard 2-(∆∆CT) method (Livak & Schmittgen, 2001),
∆∆CT = (Cttarget – Ctreference)Time X – (Cttarget – Ctreference)Time 0
Average Ct values of melanic or non‐melanic samples (Time X) were normalized using reference gene (ACTB) and depending on the calibrator sample (Time 0). The results were presented as fold change (2-(∆∆CT), i.e. as the expression ratio. Positive values of fold change indicated an upregulation of the target gene, while a negative value represents a low expression of that gene (Livak & Schmittgen, 2001). Some factors can bias the fold change of the analysis, such as the PCR efficiency or the absence of expression.
The other method used was Pfaffl method (Pfaffl, 2001), where PCR efficiencies of target and reference gene were taking into account to calculated the ratio of relative expression,
ratio ��E�������∆�������� ����������������
�E����∆����� ����������������
PCR efficiencies were calculated by performing a standard curve using six 1:10 dilution series run in triplicate for each gene (MC1R and ACTB). PCR efficiency is affected by different factors, like the specific use of PCR machine, reagents, or primers. A tail sample from a non-melanic P. lilfordi population from Rei islet (Menorca) was used to perform the standard curve for the gene expression experiment based on tail samples. In the analyses
68
Materials and Methods
Figure 36. Standard curves for target gene, MC1R (1) and reference gene, ACTB (2) based on melanic egg sample (Aire25_2). Amplification plot (3) are created when the fluorescence signal from each sample is plotted against cycle number. That plot repre-sents the accumulation of product over the duration of the real-time PCR experiment.
The samples used in this plot are a dilution series of the reference gene (ACTB).
1
2
3
4
Results
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Results