Lightcycler 480 2 platform

RNA was extracted from 70 to 80% confluent T75 flasks (about 10⁷ cells) of untreated and 5 μM IAPP treated Rin-5F cells using an RNeasy kit (Qiagen, West Sussex, UK) according to the manufacturer’s instructions. The RNA concentration and purity were measured using an Agilent 2100 Bioanalyser. The RNA purity was measured from the A260nm/A280nm ratio and was always in the range of 1.9 to 2.0. RNA was normalized for all the cell samples to 8.5 μg for the cDNA synthesis and reverse transcribed using qScript® cDNA SuperMix (Quanta Biosciences, Gaithersburg, MD, U.S.A) according to the manufacturer’s instructions. Quantitative real time PCR was performed using the Light-Cycler® 480 II platform (Roche Diagnostics, UK). The PCR was performed in 10 μl of reaction volume with 5 μl of qPCR MasterMix Plus for SYBR® Green, 4 μl of 10x diluted cDNA and 0.1 μl of each forward and reverse primer at the stock concentration of 20 μM. The mixtures were then loaded in a 384 well plate. The plates were sealed with Microseal ‘B’ film (Bio-Rad, UK) and centrifuged at 800 g for 1 min. The PCR conditions were: activation of enzymes at 95 °C for 10 min, 40 cycles of denaturation at 95 °C for 15 s, annealing and extension at 60 C for 1 min.

Quantitative real-time polymerase chain reaction (RT-qPCR) was employed to corroborate the expression patterns of selected mRNA, miRNA, lncRNA, and circRNA candidates. For each specimen, 1 μg of total RNA underwent reverse transcription to synthesize complementary DNA (cDNA) in adherence to the guidelines provided by the First Strand cDNA Synthesis Kit (Catalog #FSK-101; TOYOBO, Tokyo, Japan). The ensuing RT-qPCR amplifications were facilitated using the SYBR Green Real-time PCR Master Mix (Catalog #11201ES03; Yeasen, Shanghai, China) and executed on the LightCycler480 II platform (Roche, Basel, Switzerland). Expression quantifications were derived employing the 2^−ΔΔCt methodology, and data are delineated in terms of log2 fold change for enhanced clarity and interpretation.

Based on the results above, 25 genes and 5 lncRNAs associated with the MAPK signaling pathway were randomly selected for further quantitative determination by qRT-PCR. The RNA samples used for qRT-PCR were derived from the samples used for sequencing. One microliter of total RNA (500 ng/μL) was reverse-transcribed into cDNA using a PrimeScript™ RT Reagent kit (TaKaRa, Dalian, China). Primers were designed for each gene using the BLAST online software provided by the NCBI database and then synthesized by GENEWIZ Co. Ltd. (Tianjin, China) (Table S2). The qRT-PCR was performed on a LightCycler 480 II platform (Roche, Basel, Switzerland). A final volume of 20 μL for the qRT-PCR reaction system consisted of 10 μL of 2x SYBR Green Real-time PCR Master Mix (TaKaRa, Dalian, China), 0.8 μL of forward and reverse primers (10 μmol), 2 μL of cDNA (500 ng/μL), and 6.4 μL of RNase-free ddH₂O. The cycling conditions included an initial activation phase at 95°C for 3 min, followed by 40 cycles at 95°C for 15 s (denaturation) and at 60° ± 1°C for 15 s (annealing), with an extension phase at 72°C for 20 s. The mRNA and lncRNA abundances were calculated using the 2^−ΔΔCt method [27 (link)]. Three technical replicates were performed for each sample.

The PCRs in this study were performed on a Roche Light-Cycler 480-II platform. The HMBS-based quantitative PCR was used as an endogenous quality control to verify the quality of the DNA in the samples [21 (link)].

For in vivo biopsies obtained on different days of healing, RNA isolation, cDNA synthesis, and qRT-PCR were performed as explained previously [27 (link)]. Briefly, after tissues were diced, 0·5–1 ml Trizol was added and the tubes were introduced to Qiagen tissue lyser (Qiagen, Crawley, UK) to homogenization. Additionally, it was centrifuged and the resulting supernatant was mixed with 0·2 ml chloroform (cat. 10050090; Fisher Scientific, Loughborough, UK). The mixture was then spun to get the upper aqueous layer and mixed with an equal volume of 70% ethanol, which was then further processed with RNeasy kit (cat. 74106; Qiagen) according to the manufacturer’s instructions to extract total RNA. DNase treatment was then carried out using DNA-free kit (cat. 79254; Life Technologies, Paisley, UK) according to the manufacturer’s protocol. qScript cDNA SuperMix (cat. 95048; Quanta Biosciences, Gaithersburg, MD, USA) was used for cDNA synthesis. qRT-PCR was done in LightCycler 480 II platform (Roche Diagnostics, Burgess Hill, UK). RPL32 was used as the internal control and ΔΔC_T method was used to calculate fold change of gene expression. The primers used are detailed in S3 Table.

High-resolution melting curve analysis following PCR was performed on a Roche Light-Cycler 480-II platform as described before27 (link)28 (link). The one-step RT PCR consisted of one cycle at a 30 min reverse-transcription step at 55 °C followed by 18 high-stringency step-down thermal cycles, 40 low-stringency fluorescence acquisition cycles, and melting curve determination between 38 °C and 85 °C. The cycling parameters for FRET-qPCR were 1 × 30 min at 55 °C; 6 × 1 sec at 95 °C, 12 sec at 72 °C, 30 sec at 72 °C; 9 × 1 sec at 95 °C, 12 sec at 70 °C, 30 sec at 72 °C; 3 × 1 sec at 95 °C, 12 sec at 68 °C, 30 sec at 72 °C; 40 × 1 sec at 95 °C, 8 sec at 56 °C and fluorescence acquisition, 30 sec at 67 °C, 30 sec at 72 °C; and 1 × 38 °C to 85 °C in 2.2 °C increments with continuous fluorescence acquisition.