Taqman primer sets

Total RNA was purified from human normal, and OA synovium or cartilage as described [29 (link),30 (link)], and its quality was assessed by measurement of optical density at 260 and 280 nm. One microgram of total RNA was primed with oligo (dT) 18 primers, and complementary DNA was synthesized using a cDNA Synthesis Kit (Clontech, Mountain View, CA, USA). Predesigned TaqMan primer sets—AOC3 or VAP-1 (Hs00907292_m1) and GAPDH (Hs99999995_m1) (Thermo Fisher Scientific, Rockford, IL, USA) were used, and target (SSAO) mRNA expression was normalized to the housekeeping gene GAPDH. qRT-PCR reactions were run in an ABI Prism 7300 sequence detection system (Thermo Fisher Scientific, Rockford, IL, USA). Relative gene expression levels were calculated using the 2^∆∆Ct method [34 (link)].

Placental biopsies were ground to obtain a homogenous representative sample. RNA isolation, RT reaction, and real-time PCR were performed as previously described [41 (link)]. Samples were analyzed on the CFX Connect™ system (Bio-Rad, Hercules, CA, USA) using the QuantiTect^® Probe PCR kit (Qiagen, Hilden, Germany), and Taqman primer sets (Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer’s protocol. Negative controls were included in each run. Gene expression was determined for DHCR7 (Hs01023087_m1), CYP2R1 (Hs01379776_m1), LRP2 (Hs00189742_m1), CUBN (Hs00153607_m1), GC (Hs00167096_m1), CYP27B1 (Hs01096154_m1), CYP24A1 (Hs00167999_m1), and VDR (Hs01045840_m1). The housekeeping gene β-actin (ACTB, Hs01060665_g1) was used to normalize target mRNA levels. Quantitative analysis was performed with the aid of standard curves as previously described [41 (link)].

RNA was extracted with the RNeasy mini kit (Qiagen) following the manufacturer’s instructions. Following extraction, RNA concentration was measured by spectrophotometry (Nanodrop). The Quantitect reverse transcription (RT) kit (Qiagen) was used to transcribe 1 μg isolated RNA to cDNA.
PCR was carried out with TaqMan Fast Advanced master mix (ThermoFisher Scientific 4444963) and TaqMan primer sets (Thermo Fisher Scientific) as listed in Table 2. Negative controls included a no-RT control using the original RNA extraction as a template and a no-template control using water in place of a template. Quantitative PCR was performed in 10 µl reactions in a QuantStudio 7 thermocycler (ThermoFisher Scientific). The QuantStudio software was used to identify threshold-crossing values (Ct). The ∆∆Ct method is then used to estimate relative changes in expression level with TATA-box binding protein (TBP1) used as an endogenous control.