Validation using reverse transcription quantitative PCR (RT-qPCR)

A second method of measuring gene expression, reverse transcription quantitative PCR (RT-qPCR), was used to verify sbcC expression. In this technique, a target amplicon is

quantified by measuring fluorescence levels as the amplicon is amplified during PCR. A fast, sensitive and reproducible process, qPCR is commonly used to investigate gene expression (290). Many factors, including differences in the amounts and quality of starting material and the efficiencies of cDNA synthesis and amplification, can influence the accuracy and reliability of qPCR results (290). Therefore, qPCR data is frequently normalised against reference genes. As reference genes are essentially endogenous controls that display the same level of expression across all experimental

148 conditions, the effects of any technical variation are minimised and different samples can be compared (290). It is now common practice to use at least two genes as internal controls for the relative quantification of gene expression. The 2−ΔΔCQ _{method (291),}

first described by Livak and Schmittgen (2001), can then be used to calculate changes in gene expression. For this study, cDNA was synthesised from the RNA of the three aerobic and three anaerobic samples (section 2.2.10.3) and quantified (section 2.2.10.4). All qPCR reactions presented in this chapter were performed in triplicate and so, all values reported are mean values of the three replicates.

5.2.1.2.1Determination of reference genes

As gene expression can differ across samples and experimental conditions, the suitability of reference genes also needed to be examined. In this study, the candidate reference genes that were chosen for investigation had either previously been used for qPCR studies in other bacteria (292, 293), or were those that were deemed to have equal expression under aerobic and anaerobic conditions via RNAseq. In total, five candidate

reference genes were selected; the transcriptional regulator encoding soxR, the alkaline

phosphatase encoding phoA, the RNA polymerase sigma factor encoding rpoD, the

membrane protein encoding yegO and the pyrroline-5-carboxylate reductase encoding proC.

The three aerobic samples were pooled together in equal amounts, as were the anaerobic samples, and qPCR was performed on 10-fold serial dilutions of the two cDNA pools as detailed in section 2.2.8. Amplification data was analysed using LinRegPCR version 2013.0 (201). Amplification of each reference gene revealed a single peak in the melt curve analysis (data not shown), indicating that the primer pairs were specific to a single sequence. As an assumption of the 2−ΔΔCQ _{method is that the amplification efficiencies}

(E) of the reference genes and the gene of interest should be roughly equal (291), the efficiencies of the five candidate reference genes were considered, and found to range from 2.010 to 2.070 (Table 5.1). These results indicate that for each gene, the qPCR

amplification was 100% efficient (i.e. the PCR product was doubled with each amplification cycle). The correlation coefficients (R2) of the five candidate reference genes ranged from 0.994 to 1.000, indicating that the standard curves of the five genes had a good fit. Quantification cycle (CQ) values indicate the number ofcycles required to reach the fluorescence threshold and give an indication of the number of gene copies

149 present. In this study, the CQ values of the five reference genes ranged from 26 to 31

(Table5.1).

An ideal reference gene should have a constant level of expression, regardless of the experimental conditions (291). This expression level is represented by N0, which is proportional to the number of copies of the gene present in the sample. None of the five genes were significantly differentially expressed between aerobic and anaerobic samples (p > 0.05) as determined by Student’s t-test (Table 5.1). However, the

expression levels of phoA varied between aerobic and anaerobic samples, with

eight-fold more expression in anaerobic samples (Table 5.1). Thus, phoA was not a

suitable reference gene. Both soxR and yegO were also considered to not be appropriate

reference genes, especially for yegO which had R2 values less than the recommended

threshold value of 0.999 (Table 5.1). Thus, rpoD and proC, with almost equivalent

expression levels under both growth conditions (Table 5.1), and similar amplification

efficiencies, were chosen as the reference genes to be utilised in the relative quantitation of sbcC expression.

For the five candidate reference genes, there were discrepancies between the fold changes obtained by RNAseq and RT-qPCR, with the latter technique detecting more copies of all genes across anaerobic samples (Table 5.1); this is discussed further in

section 5.2.1.3. Potentially, the exclusion of the third anaerobic sample from RNAseq analysis, but inclusion in the RT-qPCR analysis may account for some of these differences. Nonetheless, it is difficult to explain the magnitude of the difference in expression for phoA by RT-qPCR (Table5.1).

150 Table 5.1. Summary of RT-qPCR amplification of reference genes for cDNA derived from aerobic and anaerobically grown REL4536.

soxR phoA rpoD yegO proC

RT-qPCR Aerobic CQ 28 31 26 30 28 N0 5.07 × 10-9 1.88 × 10-9 9.69 × 10-8 1.08 × 10-9 2.45 × 10-8 N0 SEM 1.04 × 10-9 4.04 × 10-10 3.02 × 10-8 5.60 × 10-11 5.50 × 10-9 R2 _{0.999 0.999 1.000 0.994 0.999} Anaerobic CQ 27 30 26 30 28 N0 9.25 × 10-9 1.52 × 10-8 1.15 × 10-7 2.05 × 10-9 3.23 × 10-8 N0 SEM 2.85 × 10-9 1.38 × 10-8 4.51 × 10-8 1.25 × 10-9 1.25 × 10-8 R2 _{0.999 0.999 0.999 0.997 1.000} Overall E ± SEM 2.07 ± 0.02 2.05 ± 0.01 2.05 ± 0.02 2.01 ± 0.02 2.03 ± 0.02 Fold change† _{1.83 8.10 1.19 1.90 1.32} p 0.240 0.388 0.749 0.517 0.598 RNAseq Fold change‡ _{-1.09 1.04 -1.06 -1.05 -1.01} p-adj 0.300 0.750 0.960 0.450 0.290

†_{Fold change for RT-qPCR data is calculated as the anaerobic N}

0 value over aerobic N0 value.

‡_{For RNAseq, fold change is calculated as the anaerobicvalue over aerobic value and negative values indicates up-regulation in}

aerobic conditions.

CQ represents quantification cycle values, R2 _{represents correlation coefficients and E represents amplification efficiencies with}

standard error of the mean (SEM) calculations. N0 is proportional to the number of copies of the gene present in the sample with

151

5.2.1.2.2sbcC gene expression verification by RT-qPCR

To study sbcC expression, equal amounts of cDNA from the three aerobic and three

anaerobic samples were amplified by qPCR as described in section 2.2.8 and analysed using LinRegPCR version 2013.0 (201). In order to use the 2−ΔΔCQ _{method (291) for the}

relative quantification of sbcC expression, rpoD and proC were also amplified by

qPCR. Amplification of sbcC revealed the presence of a single peak in the melt curve

analysis (data not shown), indicating that sbcC amplification was specific. While the

amplification efficiencies of all three genes fell within range of each other, the N0 of

anaerobic samples was almost two- and three- fold greater for the rpoD and proC genes,

respectively, than the N0 of aerobic samples. Average values of two independent runs are summarised in Table 5.2. To use the 2−ΔΔCQ method, the reference genes need to be

equally expressed under all of the experimental conditions of the assay (291). As these conditions were violated in this assay, the 2−ΔΔCQ _{method was considered to not be an}

appropriate way to quantify the relative change in sbcC gene expression under aerobic

and anaerobic conditions.

Table 5.2. Summary of RT-qPCR amplification of sbcC for cDNA derived from aerobic and anaerobically grown REL4536.

rpoD proC sbcC Aerobic CQ 22 25 28 R2 _{1.000 0.999 0.997} Anaerobic CQ 21 23 25 R2 _{0.999 0.999 0.999} E ± SEM 2.03 ± 0.01 2.00 ± 0.02 2.00 ± 0.02 N0 fold change† 1.95 3.06 19.51

†_{Fold change for RT-qPCR data is calculated as the anaerobic N}

0 value over aerobic N0 value, where N0 is proportional to the

number of copies of the gene present in the sample. CQ represents quantification cycle values, R2 _{represents correlation coefficients and E represents amplification efficiencies with}

152 Therefore, relative quantification of sbcC expression was achieved by normalisation

against the N0 of both reference genes, as detailed in Table 5.3. Using this method, sbcC expression was greater in anaerobically grown samples, however this difference in

expression was not significant (p> 0.05, Student’s t-test).

Table 5.3. Quantification of sbcC expression using rpoD and proC expression values. Aerobic 1 N0 Aerobic 2 N0 Aerobic 3 N0 Anaerobic 1 N0 Anaerobic 2 N0 Anaerobic 3 N0 rpoD 5.00 × 10-6 1.11 × 10-6 1.38 × 10-6 5.49 × 10-6 2.96 × 10-6 6.16 × 10-6 proC 6.16 × 10-7 1.89 × 10-7 4.63 × 10-7 8.58 × 10-7 3.72 × 10-7 2.65 × 10-6 sbcC 6.23 × 10-8 2.92 × 10-8 4.51 × 10-8 3.17 × 10-7 5.19 × 10-8 2.30 × 10-6 sbcC/rpoD 0.012 0.026 0.033 0.058 0.018 0.373 sbcC/proC 0.101 0.154 0.097 0.369 0.140 0.868 Aerobic mean N0 Anaerobic mean N0 Fold change† p

sbcC/rpoD 0.024 0.150 6.3 0.327

sbcC/proC 0.118 0.459 3.9 0.189

†_{Fold change for RT-qPCR data is calculated as the anaerobic N}

0 value over aerobic N0 value, where N0 is proportional to the

number of copies of the gene present in the sample.