Real time PCR

2.8.1 Preparation of reagents

All primers used in real time PCRs (polymerase chain reactions) were reconstituted using nuclease-free water under the Microflow OMNI PCR workstation (ASTEC). Primer and probe stocks were then diluted in nuclease free water to 10μM and 5μM working stocks,

respectively, and stored at -20°C in 100μl aliquots. Prior to use, the 96-well plate, adhesive film, microcentrifuge tubes and nuclease-free water were placed in a PCR UV cabinet (Bio Air Instruments) for at least 30 minutes to reduce contamination.

64 2.8.2 Standard curves

A standard curve consisting of a 10-fold serial dilution of plasmid DNA was loaded in

triplicate on each plate. A number of plasmids were engineered for the creation of standard curves for the various real time PCR assays used in this project (Table 2.5). Plasmids p.7D1 and p.7D1.β2M were engineered by Dr Helen Tuppen prior to the start of this work. A detailed protocol for the creation of plasmids p.MTCN1 and p.MTCN2 is provided in chapter six.

Plasmid Genes

p.7D1 MT-ND1, MT-ND4, MT-Dloop

p.7D1.β2M MT-ND1, MT-ND4, MT-Dloop, β2M

p.MTCN1 MT-ND1, MT-ND4, MT-Dloop, β2M, RNA18S

p.MTCN2 MT-ND1, MT-ND4, MT-Dloop, β2M, RNA18S, MT-RNR2, GAPDH

Table 2.5: Plasmids engineered for use as standard curve DNA.

Average Cq values for each standard were plotted against the dilution factor on a base-10 semi-logarithmic plot and a line of best fit applied. This graph was used to calculate the reaction efficiency using Equation 2.8:

𝐸 = 10−𝑠𝑙𝑜𝑝𝑒1

% 𝐸𝑓𝑓𝑖𝑐𝑖𝑒𝑛𝑐𝑦 = (𝐸 − 1) ∗ 100

Equation 2.8: Calculating efficiency of real-time PCR reactions. E=efficiency; slope =

gradient of standard curve.

The standard curve was accepted if R2 _{> 0.990 and the reaction efficiency was between 90-}

110% (represented by a gradient of -3.1 to -3.5). This was then used for absolute quantification of sample genes.

2.8.3 mtDNA deletion level assay

Deletion level was determined using multiplex MT-ND1/MT-ND4 Taqman® real-time PCR assay, as previously described by Krishnan et al. (2007). Primers and probes used in this protocol are detailed in Table 2.6. 5μl sample DNA were added to 15μl of mastermix,

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consisting of 10μl TaqMan® Universal Master Mix II (with UNG), 300nM forward and reverse primers for MT-ND1 and MT-ND4, 100nM molecular probes for MT-ND1 and MT-ND4 and nuclease-free water. Patient DNA samples were used at approximately 0.01ng/μl, in order to produce a Cq value between 20-30 and within the range of the standard curve. Plates were sealed with adhesive film and mixed thoroughly by vortexing for 10 seconds. The plate underwent centrifugation at 300rcf for two minutes before loading into the ABI

StepOnePlus™ Real-Time PCR System (Applied Biosystems). The reaction was carried out under the cycling conditions in Table 2.7. Data were analysed using ABI StepOne software v2.0 (Applied Biosystems).

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Target Position Sequence Tm(°C) Length (nt) %GC

Primers MT-ND1_F nt.3485-3504 5’-CCCTAAAACCCGCCACATCT-3’ 58.5 20 55 MT-ND1_R nt.3553-3532 5’-GAGCGATGGTGAGAGCTAAGGT-3’ 60.0 22 55 MT-ND4_F nt.12087–12109 5’-CCATTCTCCTCCTATCCCTCAAC-3 57.9 23 52 MT-ND4_R nt.12170–12140 5’–CACAATCTGATGTTTTGGTTAAACTATATTT-3’ 52.5 31 26 β2M _F nt.8969-8990 5′-CCAGCAGAGAATGGAAAGTCAA -3′ 55.8 22 45 β2M _R nt.9064-9037 5’-TCTCTCTCCATTCTTCAGTAAGTCAACT-3′ 57.4 28 40 Probes MT-ND1 nt.2966-2987 VIC-5’-CCATCACCCTCTACATCACCGCCC-3’-MGB 64.2 24 63 MT-ND4 nt.12111–12138 6FAM-5’-CCGACATCATTACCGGGTTTTCCTCTTG-3’-MGB 61.6 28 50 β2M nt.9006-9032 6FAM-5’-ATGTGTCTGGGTTTCATCCATCCGACA-3’-MGB 61.7 27 48

Table 2.6: Primers and probes used in standard real-time PCR protocols. F=forward, R=reverse primers; GenBank accession numbers: MT-ND1

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Process Temperature (°C) Time Cycles

UNG amperase activity 50°C 2 minutes 1 cycle

Initial denaturation 95°C 10 minutes 1 cycle

Denaturation 95°C 15 seconds

40 cycles

Primer annealing and Extension 60°C 1 minute

Table 2.7: Cycling conditions for real-time PCR reactions.

2.8.4 Determining mtDNA copy number in homogenate tissue

In homogenate skeletal muscle DNA samples, total mtDNA copy number was calculated as the ratio of the number of copies of the mitochondrial MT-ND1 gene to the single-copy nuclear gene β2M. A singleplex Taqman® real-time PCR assay was used to measure the level of β2M. 5µl sample DNA at concentration 1ng/µl were added to 15µl of mastermix

containing 10µl TaqMan® Universal Master Mix II (with UNG), 300nM forward and reverse primers for β2M (Table 2.6), 100nM molecular probes for β2M, 3mM MgCl2 and nuclease-

free water.

MT-ND1 copy number was measured in a separate reaction using sample DNA at a

concentration 1:100 that used for the β2M assay. This was necessary due to the vast difference in the abundance of these two genes in the sample. By using different sample concentrations, Cq values for both genes were comparable and within the optimum range of 20-30. This dilution factor was addressed and accounted for during the final calculations. 5µl diluted sample DNA were added to 15µl of mastermix, consisting of 10µl TaqMan® Universal Master Mix II (with UNG), 300nM forward and reverse primers for MT-ND1 (Table 2.6), 100nM molecular probes for MT-ND1 and nuclease-free water. Plates were sealed and underwent vortexing and centrifugation before the reaction was carried out as described in section 2.8.3.

Levels of MT-ND1 and β2M were measured using the standard curve method. MT-ND1 copy number was multiplied by 100 to account for the relative dilution factor against β2M. The ratio of MT-ND1:β2M was calculated to indicate the number of copies of mtDNA present per copy of the β2M gene. The β2M gene is a single copy gene and so to account for its presence on both chromosomes the ratio of MT-ND1:β2M was doubled, giving the total copies of

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mtDNA present per nucleus. It is important to note that this figure does not reflect the total mtDNA copy number per cell as skeletal muscle fibres are multinucleated.