Puralink rna mini kit

Primary human AF and NP cells from cadaveric human tissue (N = 4; Table 1) were seeded in 2% agarose gels using a silicone mold at 4.0 × 10⁶ cells/mL (1.6 × 10⁶ cells/construct) for 24 h in DCC+10%FBS media at 37°C 20%O₂ 5%CO₂, followed by 24 h in DCC+2.5% FBS at 37°C 5%CO₂ 5%O₂ with 0.00, 0.01, or 0.10 μg/mL (Masuko et al., 2007 (link)) of purified recombinant human tryptase from lung MCs (rhTryptase; Promega G5631). One quadrant of each construct was used to assessed viability (4 μM Calcein + 2 μM ethidium for 15 min at 37°C). The remaining portion of each construct was homogenized in TRIzol and mixed with 0.2 mL chloroform/mL TRIzol. This mixture was centrifuged at 12,000 × g for 15 min at 4°C to collect the aqueous upper phase and diluted 1:1 with molecular grade 70% EtOH before mRNA purification using the PuraLink RNA mini kit following manufacturer instructions (Life Technologies) for downstream cDNA synthesis with Maximus H Minus (Thermo Fisher Scientific M1662) and gene expression (qRT-PCR) for MC related chemoattractant SCF and angiogenic marker VEGFA (Table 2). Gene expression was quantified using the comparative ΔΔCt method (Livak and Schmittgen, 2001 (link)) with levels normalized to the housekeeping gene 18s (Table 2).

SCF gene expression was also analyzed in human IVD cells from autopsy for each region using qRT-PCR with 1.0 × 10⁶ cells from each group (NP, AF and EP). RNA was extracted by centrifuging the cells at 2,000 × G for 5 minutes, removal of media, and addition of 0.3 mL of Lysis Buffer (Life Technologies) with 1% 2-mercaptoethanol and an equal volume of 70% ethanol. Binding, washing, and elution was carried out using the PuraLink RNA Mini Kit as per manufacturer’s instructions (Life Technologies) and samples converted to cDNA using qScript XLT cDNA SuperMix (Quanta Biosciences). Data was analyzed using the comparative 2 delta delta Ct method, where mRNA levels were normalized to the endogenous control (18 s) and experimental controls⁷³ .