Qscript microdna cdna synthesis kit

Total RNA was extracted from lung tissue homogenates. Amplification and measurement of target RNA was performed on the Step 1 real time PCR system as previously described (Karl et al., 2006 (link)). α_v-integrin, collagen type I and TGF-β were measured using RNA extracted from lung punches or lung tissue. ER subtypes, TGF-β and IGF-1 mRNA expression was measured using RNA extracted from exosomes. The TaqMan rRNA control reagents kit (Life Technologies) was used to detect 18 S rRNA gene, an endogenous control, and samples were normalized to the 18 S transcript content as previously described (Potier et al., 2002 (link)). For miRNA analyses, cDNA was generated using qScript microDNA cDNA Synthesis Kit (Quanta Biosciences, Beverly, MA) according to manufacturer’s instructions (Elliot et al., 2019 (link)). Amplification of all miRNAs was performed on the QuantStudio 3 96well 0.2 ml Block Real-Time PCR System using specific primers, miR-let-7d, miR-29a-5p, miR-34a-5p, miR-142–3 p, miR-199a-3p, and miR-181b (IDT, Coralville, IA) using Real-Time SYBR Green qRT-PCR Amplification kit (Quanta Biosciences, Beverly, MA). U6 expression was used as a control for miRNA analyses, and relative expression was calculated using the comparative C(T) method (Schmittgen and Livak, 2008 (link)).