88 Estado nutricional y caries dental

To establish the activity of hit compounds out-with the GPF based reporter assays, their effect on IFNβ and MxA gene expression was assessed.

This was achieved through total RNA extraction of compound treated, SeV infected cells, followed by reverse transcription of messenger RNA (mRNA) and quantitative PCR (qPCR) of the resultant complementary DNA (cDNA).

2.7.1 RNA extraction

A549 cells were seeding into 6-well plates at 3x105 cells/ml (2 ml/well) and incubated at 37°C with 5% CO2. The following day, confluent cell

monolayers were treated with compound for 2 hours followed by either a 3-hour SeV infection or an 18-hour IFNα treatment. Total RNA was then extracted from

the cells by standard Phenol-Chloroform extraction using TRIzol (ThermoFisher). Briefly, 1 ml of TRIzol reagent is added to each well to lyse the

cells. Cell lysates were collected and mixed with 200 µl of chloroform. To

separate RNA, DNA and protein, the TRIZol-chloroform lysates were centrifuged at 12,000 ×g for 15 minutes at 4°C. The RNA-containing aqueous

phase was then removed from the separated lysates. RNA was then mixed with 1µl of GlycoBlue coprecipitant (ThermoFisher) and 500 µl of isopropanol (IPA)

and incubated at -20°C for up to 1 hour to aid RNA precipitation. The RNA-IPA

was then centrifuged at 12,000 ×g for 10 minutes at 4°C and the resultant RNA

pellet washed twice in 1 ml of 75% (v/v) ethanol (EtOH). The EtOH supernatant was removed from the RNA pellet, which was then air-dried and dissolved in nuclease-free water overnight at 4°C. The RNA was then used directly in cDNA

synthesis or stored at -70°C.

2.7.2 Complementary DNA synthesis

Reverse transcription of purified RNA was completed using RevertAid First Strand cDNA Synthesis Kit (ThermoFisher), which utilizes a recombinant M-MuLV reverse transcriptase. Manufacturer’s instructions were followed and are outlined below.

Total RNA (3 µg) was added to 1.5 µl of Oligo d(T)18 primer (12.5µM)

and nuclease-free water on ice and incubated at 65°C for 5 minutes. Once back

on ice, 5× reaction buffer, RiboLock RNase inhibitor (20 units), and dNTPs (1 mM of each) and RevertAid M-MuLV Reverse Transcriptase (200 units) were added to the reaction. Following an hour’s incubation at 42°C, the reaction was

terminated by a 5-minute incubation at 70°C. Resultant cDNA was used directly

2.7.3 Quantitative PCR

Quantitative PCR is a highly sensitive technique that allows accurate determination of transcript levels within a given sample. In this study, qPCR was used to compare the levels of IFNβ mRNA in samples taken from cells treated

with hit compounds and subsequently infected with SeV to activate the IFNβ

induction pathway. MxA mRNA levels in samples from cells that had been treated with hit compound and subsequently incubated with IFNα to activate the

IFN signalling pathway were also assessed. In this instance, absolute quantitation was used, as opposed to relative quantitation. To potentiate absolute quantitation, a DNA standard, of known concentration, matching the sequence to be amplified in the qPCR reaction is required. A 10-fold serial dilution of the standard allows the cycle threshold (Ct) values to be used to

construct a standard curve, from which the quantity of DNA in the test samples can be calculated. The details of this process and the qPCR are given below.

2.7.3.1 Standard curve generation

Plasmids containing gene fragments of IFNβ, MxA and β-Actin to be

amplified in the qPCR reactions were used to create a standard curve of known concentration against Ct value using a 6-point 10-fold serial dilution of DNA

from 1 ng to 0.01 pg. These reactions are carried out in the same 96-well plate as the corresponding test sample reactions, detailed below. The qPCR results for the IFNβ standard are shown in figure 2.1. In addition to the internal controls

in the qPCR reaction detailed below, the standard curve also potentiates assessment of the efficiency of the reaction through the R2 value generated (Figure 2.1A). An example of the amplification plots generated for the DNA

standards is also shown (Figure 2.1B). The dissociation curve created during each reaction displays one clear peak (Figure 2.1C), suggesting high specificity of the primers and an absence of primer dimer formation. Standard and dissociation curves for the MxA and β-Actin primer sets are shown in Appendix

1.

2.7.3.2 qPCR of test samples

Primers specific to IFNβ (nt 40-155), MxA (nt 570-931) and Actin (nt 257-

640) (Table 2.6) were used at 100 nM to assay cDNAs generated by reverse transcription (2.7.2) in qPCR reactions. In this study MESA Blue qPCR mastermIx (Eurogentec) containing SYBR Green I, an intercalating dye that fluoresces upon binding to DNA, and Rox, a reference dye that acts as an internal control to normalise any variations in mastermix concentration or reaction volume, were used. PCR reactions were set (Table 2.9) and were carried out in a Stratagene Mx3005P real-time PCR thermocycler using the specified cycling conditions (Table 2.10). Conversion of Ct value to DNA

quantity (ng) was carried out by MxPro software (Stratagene). Further data analysis was carried out on Excel (Microsoft) and Prism6 (Graphpad).

Figure 2.1: Data output of qPCR reactions using IFNβ standard template DNA.

pJet1.2 IFNβ nt 40-155 was used to create a 6-point standard curve using a 10-fold serial

dilution from 1 ng to 0.01 pg, giving an R2 value of 0.999 (A). The amplification plots for each of

the 6 DNA concentrations show even spacing, with the highest DNA concentration

corresponding to the lowest Ct value (B). The corresponding dissociation curve display a single

clean peak with no indication of primer dimer formation (C).

A C B Input DNA (ng) Ct ( dR ) Cycle F lu ore sce nce ( dR ) Temperature (°C) F lu ore sce nce (-R ’(T )) R2 _{= 0.999}

Table 2.9: Components of a typical qPCR reaction

Component Stock

Concentration

Final Concentration

MESA Blue Mastermix 2× 1×

Forward primer 100 µM 100 nM

Reverse primer 100 µM 100 nM

cDNA - 1 µl

Nuclease-free Water

Table 2.10: Cycling conditions of a typical qPCR reaction

Step Temperature

(°C)

Time

(Seconds) Function

1 – Activation 60 120 Activates Mastermix 95 600 Activates polymerase 2 – qPCR1 95 15 Denatures 50 60 Anneals 72 60 Extends2 3 – Dissociation curve 95 60 Denatures 50 30 Anneals 95 30 Denatures3

1_{Step 2 repeated through 40 cycles} 2

Fluorescence read at the end of each extension step

3

Fluorescence read at each degree between 50 and 95°C during ramp