2.9 ELISA
Levels of collagen I and III (Col I and III) were detected by using
ELISA kits (Cloud Clone Corp., China) according to the manufacturer’s
recommendations, and the absorbance at 450 nm was read. The relative Col
I and III levels were calculated according to the standard
concentration-absorbency curve.
2.10 Quantitative real-time PCR
(qRT-PCR)
Total RNA from plasma, tissues or cells
was extracted with Trizol reagent (Thermo Fisher Scientific, USA). cDNA
was synthesized by using the miRNA Reverse Transcription System kit
(TaKaRa, Japan) or 2×SYBR real-time PCR premixture (Bioteke Corp.,
China), following the manufacturer’s protocol. Real-time PCR was
performed with the StepOnePLus Real-Time PCR System. The primer
sequences and conditions for amplification are in Supplemental Table S2
and S2, respectively. U6 or β-actin was the internal reference. The
relative expression of mRNAs and miR was calculated by the
2−△△Ctmethod.