Miscript mirna pcr array for mouse fibrosis

The expression of miRNA molecules involved in fibrosis was evaluated using a miScript PCR system (Qiagen). Total RNA was isolated from liver samples using the miRNeasy mini kit (Qiagen), which ensured the effective purification of miRNA and total RNA. The concentration and purity of RNA were determined using a NanoDrop 8000 spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA). RNA was reverse-transcribed to cDNA by the miScript II RT kit (Qiagen) using a Veriti 96-Well thermal cycler (Applied Biosystems, Foster City, CA, USA). The miRNA PCR array analysis was performed by the miScript SYBR green PCR kit (Qiagen) and the miScript miRNA PCR array for mouse fibrosis (Qiagen) using a ViiA 7 real-time PCR system (Thermo Fisher Scientific, Waltham, MA, USA). The PCR array contained primers for the detection of 84 miRNA molecules involved in fibrosis and 12 internal controls. The obtained data were analyzed using the GeneGlobe data analysis software provided by Qiagen.

Total RNA, including miRNA, was isolated from primary mouse HSC using miRNeasy Mini Kit (Qiagen, Germantown, MD, USA). Mature miRNA was selectively converted into complementary DNA (cDNA) using miScript II RT Kit (Qiagen). Mature miRNA expression profiling was measured using a pathway-focused miScript miRNA PCR Array for mouse fibrosis (Qiagen). The miRNA PCR Array layout is shown in Supplemental Figure S1. The expression of miRNAs was confirmed by qRT-PCR using miScript Primer Assays (Qiagen) in a Bio-Rad CFX96 Real-Time System (Bio-Rad, Hercules, CA, USA). All procedures were conducted according to the manufacturer’s protocols.