U6 small nuclear rna snrna

Total RNA from the cells was prepared by TRIzol reagent (Invitrogen). Then, 1 μg of total RNA was subjected to reverse transcription using PrimeScript RT Master Mix (Perfect Real Time) (Takara, Shiga, Japan). Quantitative real-time PCR was carried out using the TB Green Premix Ex Taq II (Tli RNaseH Plus) (Takara) to detect FAS gene mRNA level (forward: 5’ - AGATTGTGTGATGAAGGACATGG-3’, reverse: 5’- TGTTGCTGGTGAGTGTGCATT-3’) on StepOnePlus Real-Time PCR System (Applied Biosystems, Waltham, MA, USA). GAPDH (forward: 5’-AGAAGGCTGGGGCTCATTTG-3’, reverse: 5’- AGGGGCCATCCACAGTCTTC-3’) was used as the internal reference gene.
For miRNA quantification, the Taqman advanced miRNA cDNA synthesis kit was used to implement reverse transcription following the manufacturer’s instruction (Applied Biosystems, Carlsbad, CA, USA). The Taqman microRNA reverse transcription kit was used for U6 small nuclear RNA (snRNA) to perform reverse transcription according to manufacturer’s protocol (Applied Biosystems). All probes were purchased from ThermoFisher Scientific (Waltham, MA, USA). The comparative 2^−ΔΔCT method^{18 (link)} was used to analyze the relative expression level of FAS and miR-130a-5p.

Total cell RNA, including miRNAs were isolated with an miRNeasy Mini Kit (Qiagen, Hilden, Germany). RNA concentrations were determined with the DeNovix DS-11 spectrophotometer (DeNovix Inc., Wilmington, DE, USA). cDNA was generated using the TaqMan MicroRNA Reverse Transcription Kit (Applied Biosystems, Foster City, CA, USA), and miRNA expression was quantified via real-time quantitative PCR using a TaqMan Universal PCR Master Mix (Applied Biosystems) and the BioRad CFX Connect Real-Time PCR Detection System (Bio-Rad Laboratories, Hercules, CA, USA). Specific primers for hsa-miR-130a-3p were obtained from Applied Biosystems. Expression of miR-130a was normalized to the expression of either U6 small nuclear RNA (snRNA) or miR-16-5p (Applied Biosystems), and the expression of these RNAs was similar in all treatment conditions. The data were then further normalized by setting the control values to one.