The protocol detailed below for the analysis of relative gene expression by real-time RT-PCR was based on the method described in Hoile et al., 2014 (261).
2.15.1 Complementary DNA (cDNA) Synthesis
Complementary DNA (cDNA) was synthesised by reverse transcription. 1 μl of a dNTPs mix (Promega), containing equimolar amounts of each dNTP (10 mM) and 1 μl of random nonamers (Sigma-Aldrich) at 50 μM were added to 500 ng of RNA in a total volume of 10 μl and incubated for 10 min at 70oC. Samples were then placed on wet ice for at least 1 min. A master mix containing 4
μl 5 X M-MLV reaction buffer, 1 μl M-MLV Reverse Transcriptase (200 units/μl) and 5 μl RNase-free water was prepared and added to each sample (10 μl). Samples were transferred to a Veriti Thermal Cycler (Applied Biosciences, USA) where they were incubated at 15oC for 10 min, followed by 60
min at 37oC and then 10 min at 90 oC to denature the reverse transcriptase. The newly synthesised
cDNA was diluted with RNase free water to a total volume of 100 μl and stored at -20oC.
2.15.2 Real-time RT-PCR primers
Qiagen Quantitect primer assays, which contain a mix of forward and reverse primers for a specific gene were used to measure each gene of interest by real-time RT-PCR (
Table 2.3). Upon arrival the Quantitect primers were reconstituted in 1.1 ml tris-EDTA (TE) buffer (Promega) to obtain a 10X assay solution. Qiagen does not disclose the primer sequences, however, bioinformatic information was available and all Quantitect primer assays used were designed to cross exon/exon boundaries and therefore should not amplify genomic DNA. Primers for the reference genes were obtained from Primer design again as ready-made mixes of forward and reverse primers. Reference genes are used to normalise data to constitutively expressed genes that are not affected by the experimental conditions. The reference genes were selected using geNorm analysis (see section 2.15.3.2).
Table 2.3. Human primer assays used for real-time RT-PCR.
Primer target Primer Assay
FADS1 Qiagen Hs_FADS1_2_SG Quantitect Primer Assay (QT02322621)
FADS2 Qiagen Hs_FADS2_1_SG Quantitect Primer Assay (QT00077175)
ELOVL2 Qiagen Hs_ELOVL2_1_SG Quantitect Primer Assay (QT00059017)
ELOVL4 Qiagen Hs_ELOVL4_1_SG Quantitect Primer Assay (QT00017283)
ELOVL5 Qiagen Hs_ELOVL5_1_SG Quantitect Primer Assay (QT00096334)
DNMT1 Qiagen Hs_DNMT1_1_SG Quantitect Primer Assay (QT00034335)
DNMT3a Qiagen Hs_DNMT3A_1_SG Quantitect Primer Assay (QT00090832)
DNMT3b Qiagen Hs_DNMT3B_1_SG Quantitect Primer Assay (QT00032067)
EIF4A2 Primerdesign reference gene assay (HK-SY-hu)
RPL13A Primerdesign reference gene assay (HK-SY-hu)
SDHA Primerdesign reference gene assay (HK-SY-hu)
18S rRNA Primerdesign reference gene assay (HK-SY-hu)
2.15.3 Real-time RT-PCR
cDNA was diluted to a final concentration of 5 ng/µl in RNase and DNase free water, assuming 100% conversion of RNA to cDNA. A series of six cDNA standards were made by serial dilutions of cDNA prepared from HepG2 cells representing input RNA of 15 ng, 7.5 ng, 3 ng, 1.5 ng, 0.15 ng, 0.075 ng and 0.015 ng. A master-mix for each primer set was made containing 5 µl of Quantifast SYBR green (Qiagen), 0.5 µl of each primer set and 1.5 µl of RNase and DNase free water. This was added to 3 µl of sample and standard cDNA, in duplicate, in a white 384 well plate to give a final volume of 10 µl. Each plate included two no template controls (ntcs) where the sample cDNA was replaced with 3 µl of RNase and DNase free water. Standards were always measured for all primer sets on a plate. Plates were analysed using a LightCycler® 480 system (Roche Diagnostics, Switzerland) with the following cycling conditions: 95oC for 15 min, followed by 45 cycles of 95oC for 10 s, 60oC for 30 s
and 72oC for 30 s. A melt curve step followed comprising 95oC for 15 s, 60oC for 1 min and a gradual
The melt curve analysis was performed to verify amplification of a single PCR product and absence of primer dimers on the basis that different size fragments of DNA differ in their melting points. The melting curve graphically displays the change in fluorescence when double stranded DNA dissociates into single stranded DNA when its melting point (Tm) is reached. This results in a sudden
decrease in fluorescence due to the release of SYBR green. An example of a melt curve is given in Figure 2.12.
2.15.3.1 Quantification of mRNA expression levels
Expression levels of the target genes were quantified using the relative standard curve method (270) as described below.
Standard curves were constructed for both the target and reference genes. The relative amount of each target and reference gene for each sample was then determined by interpolating from the standard curve. The value for the target gene was divided by the geometric mean of the reference genes in order to normalise the data for differences in input. An example standard curve is given in Figure 2.12.
Figure 2.12. Standard curve and melt curve for FADS2.
2.15.3.2 GeNorm analysis for selection of most suitable reference genes
A GeNorm kit (Primerdesign) was used to determine the best candidate reference genes for normalisation in real-time RT-PCR experiments. Real-time RT-PCR was carried out for a panel of six reference genes (Figure 2.13) on cDNA from a representative set of samples (six replicates) for each comparative analysis of mRNA expression. The reference genes were ranked in order of expression stability using qbase+ (Biogazelle, Belgium) real-time RT-PCR data analysis software. The software generated two graphs, the first (Figure 2.13, A) shows the average expression stability value (geNorm M) for each reference gene, starting with least stable on the left and the most stable on the right. The second graph (Figure 2.13, B) was used to determine the optimum number of reference genes to include; the graph shows sequential inclusion of each reference gene, the optimum number being when the geNorm V value drops below 0.15. The optimal number of reference targets was given as four (geNorm V < 0.15). The three reference genes with the lowest geNorm M (RPL13A, SDHA, EIF4A2) were selected. For the fourth reference gene 18S was selected instead of B2M since 18S rRNA level has been shown to remain unchanged in stimulated compared with unstimulated T-lymphocytes (271).
Figure 2.13. Graphs generated by geNorm analysis software (qbase+).
(A) Graph of average expression stability value (geNorm M) for each reference gene. (B) Graph
showing the optimum number of reference genes; found when geNorm V is less than 0.15 (green line).