Measurement of gene expression

2.2.3 RNA quantification

Extracted RNA was quantified using the Nanodrop ND2000 spectrophotometer (Thermo Fisher scientific, USA). Briefly, RNA background was measured using the elution buffer (BR5 or RNase-Free water). RNA concentration and purity were measured at a 260/230 wavelength and 230/280 wavelength, respectively. All RNA samples were processed regardless of concentration.

Belgium) and put on hold at 4°C. Another master mix containing 5x First-strand buffer (Invitrogen, USA), 0.1M of 1,4-Dithiothreitol (DTT) (Invitrogen, USA), 40 units/µL of RNAseOUT (Invitrogen, USA) and sterile endotoxin free water (Sigma Adrich, USA) was added to the samples after a brief centrifugation and incubated at 42°C for two minutes. Two hundred units of SuperScript II reverse transcriptase (Invitrogen, USA) was added to the samples and the samples further incubated at 42°C for 50 minutes, and then the reaction was inactivated at 70°C for 20 minutes.

Samples were diluted to a final concentration of ±1ng/µL by adding 80µL of sterile endotoxin free water.

Each cDNA sample is split into 96 microchambers of a 96.96 Fluidigm gene Expression (GE) Array chip (Fluidigm, USA) to mix with each of the 96 primer-probe assays, consequently allowing 96 separate qRT-PCR reactions from each cDNA sample. The Fluidigm 96.96 GE chip technology requires sufficient cDNA template in each of the 96 microchambers to ensure optimal amplification of each primer-probe set (Dominguez et al. 2013). Thus, to ensure consistent distribution of cDNA template into each of the 96 microchambers, a preamplification of synthesized cDNA using a pool of the relevant primer-probe sets of interest is necessary before loading samples on the Fluidigm GE chip. Hence, transcripts of interest were amplified by conventional PCR using a mix of 2x PCR master mix (Thermo Fisher Scientific, USA) and 0.2x of the primer probe master mix of the TaqMan primer probe assays of interest (Thermo Fisher Scientific, USA). Final volume of 10µL of the mix was added in new 96-well plates and plates were sealed with strips of dome caps. The thermocycler was set to the following parameters for the PCR: 95°C for 10 minutes, 95°C for 15 seconds and 60°C for four minutes. Even distribution of mRNA in every

chamber requires at least 4,906 amplicons from 12 preamplification cycles (Dominguez et al. 2013). Therefore, steps two and three were run sixteen times (16 cycles) before the reaction was put on hold at 4°C. Samples were diluted 1:25 with endotoxin free water by adding 240µL per well. The samples generated from this step will henceforth be referred to as pre-amplified cDNA.

A confirmatory RT-PCR was performed using one of the reference primers to ensure that the samples were pre-amplified and that the reagents and the negative controls were not contaminated. The confirmatory RT-PCR consisted of two samples of interest, duplicate NTC and NoRT, and was run on the Rotor-Gene 6000 series RT- PCR instrument (Corbett life science). The RT-PCR was run using the following setting: 50°C for two minutes, 95°C for 10 minutes and for 40 cycles of 95°C for 15 seconds and 60°C for one minute. Preamplification of cDNA was repeated if amplification was detected in the NTC and/or NoRT samples, indicating a false positive result or contamination. If either the NTC or NoRT yielded amplified product a second time suggesting that the contamination occurred during cDNA synthesis, the assay ws repeated from the cDNA synthesis step. If the samples selected for the confirmatory RT-PCR were not amplified in addition to both controls, the confirmation RT-PCR was repeated to ensure that samples were amplified.

2.3.2 Fluidigm assay

The Fluidigm Biomark HD instrument was used to perform microfluidic measurement of gene expression in the amplified cDNA samples. Transcripts of interest were measured using 96.96 Dynamic GE Array chips (Figure 4A), allowing 9,216 simultaneous qRT-PCR reactions by quantifying abundance of 96 mRNA transcripts

in 96 samples. Samples and controls were run in duplicate, thus incorporating 45 amplified cDNA samples, an internal positive control (IPC), a NoRT and NTC. The IPC was pooled from different previously pre-amplified samples (using our primer- probes of interest) with varying levels of gene expression and was used to affirm that gene expression between the different experiments in each cohort (Chapters 4 and 5) was comparable. To prepare a chip for sample transfer, it was first injected with a control fluid into each accumulator and primed using the integrated fluidic circuit controllers. Assay loading reagent (Fluidigm, USA) was added on to the primer- probe assays at a 1:2 dilution, centrifuged and stored at 4°C until ready to load the chip. The samples were prepared by adding 2x TaqMan universal PCR master mix (Applied Biosystems, USA) and 20x GE sample loading reagent to 5.4µL of preAmped cDNA. Upon completion of priming, assays were loaded into the assay inlets in singlet and samples loaded into the sample inlets in duplicate (Figure 4A).

The chips were returned to the integrated fluidic circuit controller for loading and mixing of samples and assays, after which the chips were loaded into the Fluidigm Biomark HD machine. Runs were performed using the Biomark data collection software (Fluidigm, USA), and pre-analysed using the Fluidigm real-time PCR analysis software v3.1.3 (Fluidigm, USA). The quality threshold, baseline correction and Ct threshold method were set to 0.65, linear derivative and auto global normalisation, respectively. Comma separated value (.csv) files of the data were exported for further analysis.

Figure 4: 96.96 dynamic array gene expression Fluidigm chip. (A) Layout and design of the 96.96 array chip for gene expression measurement. (B) Representative heatmap of gene expression measured from a 96.96 array chip, non-template control (NTC), no reverse transcriptase (NoRT) and internal positive control (IPC) shown.

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96 Assay

inlets 96 sample

inlets 9,216 total

reaction chambers

Accumulator A

Assays

Samples

NTC NoRT IPC B