Iscript advanced cdna synthesis

RT-PCR. MirVana miRNA isolation kit (Ambion, Austin, TX, USA) was used for total RNA extraction from keratinocytes, The amount of 1 μg total RNA has been used for reverse transcription using the IScript Advanced CDNA synthesis (Biorad, code 1725037) while real-time PCR was performed using SSOADV UNIVER SYBR GRN qPCR Master Mix (Biorad, code 1725270). The expression of genes involved in the psoriasis pathway was detected using the PSORIASIS T1-4 H384 plates on a CFX384 Touch Real-Time PCR System (Biorad, code 10038559, and 1855485). All procedures were performed using the manufacturer’s instructions. Each gene expression profile was defined from the threshold cycle (Ct), and relative expression levels were calculated by using the run-file and software provided by the manufacturer (Biorad, Hercules, CA, USA), by using the internal controls and statistical tools provided. (Run file and primer validation data can be found on: https://www.bio-rad.com/it-it/prime-pcr-assays/predesigned-plate/sybr-green-psoriasis-h384).
Pro-inflammatory cytokine gene analysis in HUVEC cells, was performed by RT Profiler PCR Array System (1022A, Bioscience Corporation) according to the manufacture’s instruction.

A complete list of primers used for qPCR can be found in Supplementary Table 1. Total RNA was extracted from dissected retinas using Trizol (10296010; Invitrogen, Grand Island, NY) using standard manufacturer’s recommendations. RNA quality of RIN 7 was confirmed using Agilent High Sensitivity RNA screen Tape. cDNA was synthesized using 500 ng of total RNA per biological sample using iScript Advanced cDNA Synthesis (1725037; Bio-Rad, Hercules, CA). Quantitative PCR was performed on 1 µl of cDNA per biological sample using iTaq Universal SYBR Green Supermix (1725121, Bio-Rad) and a Bio-Rad CFX Real-Time System. Mouse Tctn1 exon2 was targeted using primers spanning the Tctn1 exon 2/exon 3 junction. To determine Tctn1 mRNA levels we generated a standard curve of known concentrations of full-length mouse Tctn1 plasmid (10 ng/µl–0.0001 pg/µl) and approximate Tctn1 mRNA levels were calculated by plotting the averaged Cq values from each biological samples to the standard curve.

RNA was extracted from testis using the RNeasy Plus mini kit from Qiagen (Hilden, Germany) following the manufacturer’s instructions. A cDNA library was produced from 2 μg of total RNA with the iScript Advanced cDNA Synthesis (Bio-rad, Hercules, CA, USA). RT-PCR was performed in a CFX96 Real-Time PCR Detection System thermocycler (Bio-Rad, Hercules, CA, USA) and analyzed with Bio-Rad CFX Manager software (Bio-Rad, Hercules, CA, USA). p21 and the housekeeping gene Gapdh primers were obtained from Biorad (Bio-Rad, Hercules, CA, USA). All samples were run in triplicate, and non-template controls were included in the analysis.