Ds 11 series spectrophotometer fluorometer

Total RNA was obtained by washing cells twice in ice-cold PBS and extracted using a miRNeasy Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer’s protocol. RNA was quantified using a DS-11 Series Spectrophotometer/Fluorometer (DeNovix, Wilmington, DE, USA). One microgram of total RNA was pretreated with RNAse free-DNAse I (Invitrogen, Waltham, MA, USA) and reverse-transcribed to cDNA with iScript Reverse Transcription Supermix (Bio-Rad, Hercules, CA, USA) according to the manufacturer’s instructions. For qPCR amplification, 5 ng of cDNA was amplified with PCSK5 or HPRT1 TaqMan probes (Thermo Fisher, Waltham, MA, USA) using TaqMan Fast Advanced Master Mix (Thermo Fisher, Waltham, MA, USA). The PCRs s were amplified in a QuantStudio 6 Flex Real-Time PCR for 40 cycles. After incubation at 95 °C for 20 s to activate Taq polymerase, the samples were amplified for 40 cycles with a 1 s denaturation step at 95 °C and a 20 s annealing/elongation step at 60 °C. The fold change in PCSK5 mRNA levels was calculated with the comparative Ct method and normalized to HPRT1.

Gene expression was analyzed by real-time quantitative PCR. The quality of RNA extracted using the High Pure RNA Isolation Kit (Roche Applied Science) was analyzed by Nanodrop spectrometry using a DS-11 Series Spectrophotometer/Fluorometer (DeNovix). RNA levels for human IFNB1, CXCL10, TNFA, A20, IL6, and GAPDH were analyzed using TaqMan® RNA-to-CT ™ 1-Step Kit (Applied Biosystems) according to manufacturer’s instructions. GAPD was used as a housekeeping gene.

In order to identify the amplified DNA fragments, 1% agarose gel electrophoresis (Bi Tools, Madrid, Spain) with the addition of the SimplySafe nucleic acid intercalator (5 μg/mL) (EURx, Gdańsk, Poland) was used. GeneRuler ™ 1 kb DNA Ladder (Thermo Fisher Scientific ™, Waltham, MA, USA) was used as the marker. Purification of PCR products from unattached nucleotides and primers was performed using the GeneMATRIX Agarose-Out DNA Purification Kit (EurX, Gdańsk, Poland) according to the manufacturer’s instructions. The samples were suspended in molecular water (Mili-Q). The purified DNA fragments were stored at −20 °C. The samples were then analysed qualitatively and quantitatively with the DS-11 Series Spectrophotometer/Fluorometer (DeNovix, Wilmington, DE, USA), and then samples were subjected to Sanger sequencing. The templates for the reaction were previously purified PCR products that were diluted according to the manufacturer’s instructions to a final concentration of 5 ng/µL with molecular water (Mili-Q). The sequencing reaction was performed by Eurofins Genomics (Ebersberg, Germany).