The expression levels of microRNAs were evaluated in serum and plasma samples. Quantitative real-time PCR assays were performed with miRCURY LNA™ Universal RT microRNA PCR with SYBR® and LNA™ primers set (Exiqon). Five micro RNAs (miR-16, miR-93, miR-423, cel-miR-39-3p, and SNORD44) were selected from the literature [58 (link),59 (link)] and used for normalization upon demonstration of stable expression among the samples. Spike-in RNAs were used to calibrate the results (Exiqon).
The expression levels of COL1A1 and NPPB were also evaluated by RT-qPCR. COL1A1 expression was evaluated using SYBR™ Green PCR Master Mix (ThermoFisher Scientific) with the following primers: (forward 5’-GTGCGATGACGTGATCTGTGA–3’; reverse 5’-CGGTGGTTTCTTGGTCGGT-3’). GAPDH was used for normalization (forward 5’-ACAACTTTGGTATCGTGGAAGG-3’; reverse 5’-GCCATCACGCCACAGTTTC-3’). NPPB mRNA amplification was conducted using the Taqman™ Universal PCR Master mix and Taqman probe Hs00173590_m1. Two references genes were tested for normalization: GAPDH (Hs02786624_g1) and HPRT (Hs02800695_m1) (ThermoFisher Scientific).
The expression levels of COL1A1 and NPPB were also evaluated by RT-qPCR. COL1A1 expression was evaluated using SYBR™ Green PCR Master Mix (ThermoFisher Scientific) with the following primers: (forward 5’-GTGCGATGACGTGATCTGTGA–3’; reverse 5’-CGGTGGTTTCTTGGTCGGT-3’). GAPDH was used for normalization (forward 5’-ACAACTTTGGTATCGTGGAAGG-3’; reverse 5’-GCCATCACGCCACAGTTTC-3’). NPPB mRNA amplification was conducted using the Taqman™ Universal PCR Master mix and Taqman probe Hs00173590_m1. Two references genes were tested for normalization: GAPDH (Hs02786624_g1) and HPRT (Hs02800695_m1) (ThermoFisher Scientific).