Mir 140 3p

TaqMan probes and primers specific to PLOD1 (P/N: Hs00609363_m1; Applied Biosystems), which are assay‐on‐demand gene expression products, were used to analyze PLOD1 expression. miR‐140‐5p (P/N:001187; Applied Biosystems) and miR‐140‐3p (P/N:002234; Applied Biosystems) expression was analyzed by qRT‐PCR. mRNA and miRNA expression levels were normalized to those of GUSB (P/N: Hs99999908_m1; Applied Biosystems) and RNU48 (assay ID: 001006; Applied Biosystems). PCR quantification was performed as described previously (Yamada et al., 2018d).

Nine miRNA were chosen for RT-qPCR validation; miR-29a-3p (TaqMan® ID 007600_mat, Applied Biosystems), miR-99a-5p (TaqMan® ID 006254_mat), miR-126-3p (TaqMan® ID 008451_mat), miR-140-3p (TaqMan® ID 471823_mat), miR-222-3p (TaqMan® ID 000525), miR-223-3p (TaqMan® ID 002295), miR-204-5p (TaqMan® ID 000508), miR-409-3p (TaqMan® ID 002332), miR-6119-5p (Custom TaqMan® small RNA Assay). Reverse transcription was achieved on 10 ng of total RNA using the TaqMan® MicroRNA Reverse Transcription (Applied Biosystems, Foster City, CA, USA) kit following the manufacturer’s instructions. In the thermal cycler (StepOne+, Applied Biosystems, Foster City, CA, USA), each 15 μL RT reaction followed 30 min at 16°C, 30 min at 42°C, 5 min at 85°C. Then, 1.3 μL of miRNA-specific cDNA from the reaction were amplified using the TaqMan® Small RNA Assays (Applied Biosystems, Foster City, CA, USA) following the manufacturer’s instructions. Amplification was performed at 95°C for 10 min, pursued by 40 cycles of 95°C for 15 s and 60°C for 1 min. All miRNA levels were normalized to the values of U6 snoRNA [47 (link),48 (link)].