qPCR analysis

2.2.25.1Total RNA isolation from whole brain

Immediately following collection of the brain, it was disrupted, quartered and snap frozen in tubes in a dry ice/ethanol bath. Samples were stored at -80°C until required. RNA was isolated using the Promega Total RNA Isolation System. Briefly, samples were homogenised in 1 mL RNA lysis buffer in 1 mL glass tissue grinders. This was divided into 175 μL aliquots and the RNA was isolated as per the manufacturer’s instructions with the optional DNase treatment. A further 2 hour DNase treatment was performed on the eluted RNA as described in Section 2.2.25.3, prior to the precipitation of RNA as described for DNA in Section 2.2.2.7. The yield of RNA was then assessed using the Qubit fluorometer as described in Section 2.2.7.

2.2.25.2Total RNA isolation from pooled DRG

Immediately following collection of DRG from spinal levels T13 to T8 they were snap frozen in tubes in a dry ice/ethanol bath. Samples were stored at -80°C until required. RNA was isolated using the RNAqueous micro kit (Ambion). Briefly, DRG were homogenised in 100 μL of the lysis solution that contains guanidinium thiocyanate using a 1 mL glass tissue grinder on ice. RNA was then isolated from the lysate as per the manufacturer’s instructions with the optional post elution DNase treatment.

As this DNase treatment was not sufficient to eliminate all viral DNA from the RNA samples, a further DNase treatment step was performed for 1 h as described in Section 2.2.25.3. Given the very small quantity of RNA (less than 1 μg) that is extracted from these

samples, routine quantification of RNA and assessment of RNA quality by gel electrophoresis was not possible.

2.2.25.3Additional DNase treatment of RNA to eliminate residual DNA

To completely eliminate DNA from the extracted RNA such that it was undetected by qPCR, a more robust DNase treatment step was required. A reaction mix was prepared containing approximately 0.1 μg RNA, 1× DNase incubation buffer (Roche), 20 U DNase (Roche), 20 U RNasin ribonuclease inhibitor (Promega) and nuclease free water (Ambion) to 30 μL. This reaction was incubated at 37°C for one to four hours, depending upon the quantity of RNA used. The enzyme was then heat inactivated by incubation at 75°C for 10 minutes.

2.2.25.4cDNA synthesis

cDNA synthesis reactions were performed using the SuperScript VILO cDNA synthesis kit (Invitrogen) according to the manufacturer’s instructions. This reverse transcriptase kit uses the SuperScript III reverse transcriptase and a random primer mix, which results in the high cDNA yields (Ståhlberg et al., 2004b). Briefly, 2 μL of RNA was mixed with 1× VILO reaction mix, 1× SuperScript enzyme mix, and made up to 20 μL with nuclease free water (Ambion). This was then incubated at 25°C for 10 minutes, followed by incubation at 42°C for 60 minutes. The reaction was terminated by incubation at 85°C for five minutes and stored at -20°C until it was required.

2.2.25.5Construction of RNA standards for qPCR assay

Plasmid DNA was isolated and linearised with either HindIII or NotI (refer to Sections 2.2.2.1 and 2.2.4). The resulting DNA fragment was analysed by agarose gel electrophoresis to confirm that the plasmid was fully linearised to prevent long run-on transcripts with multiple copies of the transcript in the same message (refer to Section 2.2.5). To synthesise RNA, 2 μg of linearised plasmid DNA was mixed with 1× transcription optimised reaction buffer (Promega), 10 mM DTT (Promega), 100 U RNasin ribonuclease inhibitor, rNTP mix (500 μM each; Promega), and 40 U SP6 RNA polymerase (Promega). This mix was then incubated at 37°C for two hours. To eliminate plasmid DNA, a DNase treatment was performed as described in Section 2.2.25.3 for four hours. The RNA concentration was then quantified using the Qubit fluorimeter as described in Section 2.2.7.

The RNA was then diluted in ten-fold serial dilutions to the desired concentration for each step of the standard curve (Table 2-13). Using each dilution and 311 ng irrelevant RNA

(refer toSection 2.2.25.1), a series of cDNA synthesis reactions were carried out according to section 2.2.25.4. These standards were then diluted one in five in nuclease free water and 2 μL of each standard was then used in each qPCR reaction.

2.2.25.6Details of qPCR assay design

All gene expression assays were designed by and purchased from Applied Biosystems. They were already optimised and further investigation of annealing temperature was not performed. The rbfox3 assay was a predesigned assay (assay ID: Mm01248771_m1; UniGene: Mm.341103) and the primer and probe sequence information is not disclosed. The probe was conjugated to the fluorescent reporter molecule VIC.

To design the Cre assay, the sequence of the eGFP/Cre fusion gene based on the plasmid pIGCN21 was supplied to Applied Biosystems. This assay was also supplied as a predesigned assay (CRE_RECOM; assay ID: A17MR8), and the probe with conjugated to the fluorescent reporter molecule 6-carboxyfluorescein (6-FAM). The details of this assay are found in Table 2-14.

For the detection of US12 transcripts (for simplicities sake, now referred to as ICP47 transcripts), previously published primer and probe sequences were selected (Table 2-14; Ma et al., 2014). These probes were conjugated 5’ to the fluorescent reporter molecule 6- FAM, with a 3’ Minor Groove Binder Non Fluorescent Quencher (MGBNFQ). The MGB

Input RNA concentration for cDNA synthesis (copies/μL)

Final copy number per qPCR reaction (copies) 2.5 × 107 _{1 × 10}6 2.5 × 106 _{1 × 10}5 2.5 × 105 _{1 × 10}4 2.5 × 104 _{1 × 10}3 1.25 × 104 _{5 × 10}2 6.25 × 103 2.5 × 102 2.5 × 103 _{1 × 10}2

Table 2-13. RNA copy number for qPCR standards. The required input RNA

concentration for each cDNA synthesis reaction to construct standards with the final copy number per qPCR reaction shown.

moiety allows more stable binding of the probe to DNA, increasing the melting temperature of the probe (Kutyavin et al., 2000).

Assay Primer/ _probe Use Concentration _(nM) Sequence

CRE_ RECOM

Forward primer

Forward primer used to

amplify eGFP/Cre 900

CGGCGGATCCGA AAAGAAAA Reverse

primer

Reverse primer used to

amplify eGFP/Cre 900

ACGCTAGAGCCT GTTTTGCA

Probe Hydrolysis probe that binds with the amplified

eGFP/Cre region 250 6-FAM- TTCACCGGCATC AACG-MGBNFQ ICP47 Forward

primer Forward primer used to amplify US12 650

GTGCACGGCGGT TCTG Reverse

primer Reverse primer used to amplify US12 650

CGTACGCGATGA GATCAATAAAAG

G Probe Hydrolysis probe that binds with the amplified

US12 region

250 CCGCCTCCCGGT6-FAM-

CCT-MGBNFQ

2.2.25.7qPCR analysis of viral transcripts within DRG

When setting up all qPCR experiments, a unidirectional workflow pattern (pre- to post- qPCR) was enforced, with physically separate laboratories utilised for the construction of mastermixes, isolation of RNA from tissues and subsequent processing, and the isolation and handling of plasmid DNA. In order to carry out absolute quantification of viral transcripts within DRG, the following experiment was carried out. The samples from each mouse were used in two assays to detect either Cre transcripts or US12 transcripts (which will be referred to as ICP47 transcripts hereafter) within the same run. Each reaction was performed in triplicate. The entire standard curve (as described in Section 2.2.25.5) was used in parallel on each plate. Each qPCR reaction to detect Cre transcripts contained 1× LightCycler 480 probes master mix (Roche), 2× rbfox3 gene expression assay, 1× Cre gene expression assay, and 3 μL of cDNA in a final volume of 20 μL. Each qPCR reaction to detect ICP47 transcripts contained 1× LightCycler 480 probes master mix, 2× rbfox3 gene expression assay, 250 nM ICP47 probe, 650 nM each of the ICP47 forward and reverse primers, and 3 μL of cDNA in a final volume of 20 μL. No template controls were included

Table 2-14. Details of oligodeoxynucleotides used for qPCR analysis. The actual Tm of the probe is unknown due to the addition of the MGB moiety, though an estimate based on the TK-specific sequence is indicated.

for all assays on each run. The qPCR assays were performed with the Roche LightCycler480, using the following program:

Preincubation - 95°C for 10 min

Amplification and detection – 50 cycles of: a. Denaturation - 95°C for 15 s b. Annealing - 60°C for 30 s c. Extension - 72°C for 1 s

Cooling – 40°C for 10 s

Threshold cycle (CT) values were calculated using Roche LightCycler 480 software (version 1.5).