Oligo dt

Aliquots of RNA samples were converted to cDNA with SuperScript reverse transcriptase and oligo(dT) (Life Technologies, Australia). Real-time PCR was performed on a Corbett Research Rotor-Gene 6000 cycler with the QuantiFast SYBR Green PCR Kit (Qiagen, Australia) as previously described [4 (link)]. Thermocycling was initiated with a 5-min incubation at 95 °C, followed by 45 cycles (95 °C for 10 s; 60 °C for 30 s). The specificity of amplification was confirmed by high-resolution melt curve analysis at the end of each run. The efficiency of each primer set (see Table S4 for primer sequences) was evaluated by standard curves using serial dilutions of cDNA samples. Each reaction was carried out in duplicate (technical repeat) with non-reverse-transcribed cDNA (RT^–) as negative controls (non-template control). Four housekeeping gene candidates, elongation factor 1-a, actin, α-tubulin and γ-tubulin were tested for stability in scion stem tissue as previously described [4 (link)]. The expression of each gene was an average of three biological replicates.

Total RNA extraction was performed with the Qiagen RNeasy Kit (Qiagen, Canada) according to the manufacturer's protocol. 1 μg of RNA was reverse transcribed using oligo-dT and Superscript II (Life Technologies, Grand Island, NY, USA) according to the manufacturer's protocol. 1 μL of the resulting cDNA mixture was added to the Platinum SYBR Green qPCR SuperMix-UDG with Rox (Life Technologies, Grand Island, NY, USA) and amplified with target gene-specific primers on the Applied Biosystems 7900HT (Carlsbad, CA; The Applied Genomics Centre, Edmonton, Alberta, Canada). All genes of interest were normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) transcript expression levels. Please see Supplementary Table 1 for list of primer sequences.

Differences in mRNA expression of IL-1β, TNF-α, GAL-1, GAL-3, MMP-3, collagen type I, collagen type III and elastin genes were analysed by qPCR in primary human fibroblasts (SK0355 and SK0235) exposed or not to activated U937 CM treated with HA, CTL and their mixture for 4, 10 and 24 h.
Total RNA was extracted using TRIzol (Life Technologies, Carlsbad, CA, USA) according to the manufacturer’s instructions. RNA quality was monitored using Nanodrop 2000c spectrophotometer (Thermo Scientific) by measuring absorbance at 260/280 nm. Total RNA was incubated with DNAse I (ThermoFisher) for 15 min to remove DNA from the samples. In total, 500 ng of total RNA was reversely transcribed using Oligo-dT and Superscript II (Life Technologies, Carlsbad, CA, USA) and qPCR was carried out using Xpert quick SYBR Green (GRISP, Portugal) on a Rotor-Gene RG -3000A (QIAGEN, Germany) [20 (link),21 (link)]. Primers used for q-PCR analysis are listed in Table 1. Gene expression was assessed using the 2ΔCt method, in which ΔCt = Ct peptidylprolyl isomerase A (PPIA)-Ct target gene.

Total RNA was extracted from stem tissues of breadfruit plants by using RNeasy kit RNeasy kit (Qiagen, VIC, Australia) and reverse transcribed with reverse transcriptase and oligo(dT) (Life Technologies, VIC, Australia). The resulting cDNA was subjected to degenerate PCR using primers 5′-ATGGAYGARYTIYTIGCNG-3′ and 5′-GCNCAYTTYACNGCNAAYCARGCN-3′ as previously described [30 (link)]. The PCR reactions were performed at 35 cycles with annealing temperature at 52 °C. The PCR products were cloned into pGEMT vector (Promega, NSW, Australia) and sequenced. Full-length cDNA clones of AaDELLA genes were amplified with Advantage DNA polymerase (Takara Clontech, CA, USA). The thermocycling was initiated with a 2-min incubation at 92 °C, followed by 32 cycles (92 °C for 30 s and 68 °C for 2 min) and a final extension of 7 min at 68 °C. The gene-specific primers were 5′-GAAAAAGATCAGAAGAAGAAGAATCATCATG-3′ and 5′-GACCCGACTTAGCGAGCCACAG-3′ for AaDELLA1 and were 5′-GTGTTTGAGGAAAAAGAGGGCCTGTG-3′ and 5′-GGGTCCGGCCCGACTCAGAG-3′ for AaDELLA2. These primers were designed from the sequence information of AaDELLA genes isolated from the “Mason” breadfruit [30 (link)]. PCR products were cloned into pGEMT vector and sequenced as above. The resulting sequences were analysed by Sequencher (version 5.4.1, Gene Codes Corporation, Ann Arbor, MI, USA).