Transcriptor universal cdna master mix

RASFs were transfected with a mixture of siRNAs using Lipofectamine® 3000 (Invitrogen) according to the manufacturer's protocol. To maximize the knockdown efficiency for the calcium binding protein (calmodulin, calpains, and calcineurin), the siRNA of these gene isoforms were mixed together before the transfection: siRNA CALM1 + CALM2 + CALM3 for calmodulin, siRNA CAPN1 + CAPN2 for calpains, and siRNA PPP3CA + PPP3CB + PPP3CC for calcineurin. All siRNA were purchased from Qiagen (Cambridge, USA).
RNA was extracted from the (AIA) animal joint tissues or immortalized RASFs using FavorPrep™ Blood/Cultured Cell Total RNA Purification Mini Kit (Favorgen Biotech Corp.). RNA concentration was determined using the NanoDrop 2000c Spectrophotometer (Thermo Scientific) and 1 μg of total RNA was used to synthesize the corresponding cDNA using the Transcriptor Universal cDNA Master mix (Roche, USA).

Total RNA was extracted from Min6 cells using RNeasy Plus mini kit and from pancreatic islets using RNeasy Plus micro kit (Qiagen, ref. 74034) following the manufacturer’s instruction. Total RNA sample concentrations were determined using a Nanodrop spectrophotometer (Implen). 100 ng of RNA was used for reverse transcription (RT) using Transcriptor universal cDNA master mix (Roche) for Min6 cells or superscript III enzyme (Invitrogen) for mouse pancreatic islets according to the manufacturer’s instructions. Gene expression was measured through quantitative real-time PCR (qPCR) using FastStart SYBR Green master mix (Roche) according to the manufacturer’s recommendations in a LightCycler 480 II device (Roche). Mouse RT-qPCR results were normalized to endogenous cyclophilin reference mRNA levels. The results are expressed as the relative mRNA level of a specific gene expression using the formula 2^−ΔΔCt.

Total RNA was extracted from the cells using a miRNeasy Micro Kit (Qiagen, Valencia, CA, USA) according to the manufacturer’s instructions. The samples were reverse-transcribed using Transcriptor Universal cDNA Master Mix (Roche, Basel, Switzerland), and RT-qPCR was performed using the LightCycler 480 SYBR Green I Master Mix (Roche) and LightCycler 96 Real-Time PCR System (Roche) in 96-well plates. The thermocycling conditions were as follows: (1) Pre-denaturation at 95°C for 300 s; (2) cycle reaction (40 times) at 95°C for 10 s, at 60°C for 20 s, and at 72°C for 6 s; and melting curve: 95°C for 5 s, 65°C for 60 s, and 97°C for 4 s. Amplification curves were analyzed using Roche LC 96 software v. 1.1, and the analysis of relative gene expression data were calculated using the 2−ΔΔCq method.⁷⁰ (link) All reactions were performed in duplicate. The results were normalized to β-actin expression levels.
The forward and reverse primers used for each gene were as follows: MIF, 5′- ACAGCATCGGCAAGATCGG-3′ and 5′- TAATAGTTGATGTAGACCCTGTCCG-3′; CDH1, 5′-TCACAGCAGAACTAACACACGG-3′ and 5′-TCAAAATGATAGATTCTTGGGTTGGG-3′; VIM, 5′-GGACCAGCTAACCAACGACA-3′ and 5′-TCCTCCTGCAATCCCG-3′; and β-actin, 5′-CCTGGCACCCAGCACAAT-3′ and 5′-GCCGATCCACACGGAGTACT-3′.

Total RNA from embryos was extracted using the TRIzol reagent (Invitrogen, Carlsbad, CA), following the provided protocol. RNA was quantified using the Nanodrop 8000 (Thermo Scientific, Waltham, MA) and its quality was checked using the 2100 Bioanalyzer (Agilent Technologies). cDNA was synthesized using Transcriptor Universal cDNA Master Mix (Roche, Basel, Switzerland). Primers pair GGCAAACAAGTCCTACAAGCT and GAGCTCCACCAGATCCAAAG amplified the sequence between exons 1 and 5. Primers specific for β‐actin were used as a positive control. To test the efficacy of the splice‐blocking morpholino, primer pairs GGCAAACAAGTCCTACAAGCT and TGCAGGTCCTCTTTCAAACTC were used to amplify a region between exons 1 and 3.