Qrt pcr trizol

Cells were washed with PBS and were then xed with 10% formalin for 30 min, after which they were washed using 60% isopropanol prior to staining for 10 minutes with Oil red O (0.3%; Sigma-Aldrich) while shaking gently. Cells were then washed using distilled water to remove free dye. In addition, free dye was eluted from these cells using 100% isopropanol, after which absorbance at 490 nm was assessed via spectrophotometry [31] .
qRT-PCR TRIzol (Invitrogen) was used to extract cellular RNA as above, and a cDNA Reverse Transcription Kit (Thermo, CA, USA) was then utilized for cDNA preparation. All qRT-PCR reactions were conducted using a SYBR Premix Ex Taq kit (Toyobo, Osaka, Japan) with an ABI Prism 7500 instrument (Applied Biosystems). The primers were used in this study are compiled in Table S1 (Supplementary Files).
Relative gene expression was quanti ed using the 2 -ΔΔCt method [32] [33] .

A Dual-Luciferase reporter assay was conducted based upon provided instructions (Promega, WI, USA). Brie y, HEK-293T cells were transduced with a lentivirus encoding miR-382-5p or a control construct and were then plated in 96-well plates until 70% con uent. After an additional 12 h, cells were co-transfected with 50 ng of pMIR-YBX1-3'UTR-wt or pMIR-YBX1-3'UTR-mut and 10ng of pMIR-GLO (Gene Pharma). After a further 24 h incubation, the Dual-Luciferase reporter assay System was used to quantify re y and Renilla luciferase activity.
qRT-PCR TRIzol (Invitrogen, USA) was used to extract cellular RNA, after which a PrimeScript RT reagent kit (TaKaRa, Japan) was utilized to prepare cDNA, while a PrimeScript miRNA cDNA Synthesis Kit (TaKaRa) was instead used for miRNA analyses. SYBR Premix Ex Taq I was then used for qPCR analyses, and all primers used in this study are compiled in table 2. β-actin and U6 served as normalization controls for mRNA and miRNA analyses, respectively, with the 2-ΔΔ Ct approach being used to normalize gene expression.

Cells were washed with PBS and were then xed with 10% formalin for 30 min, after which they were washed using 60% isopropanol prior to staining for 10 minutes with Oil red O (0.3%; Sigma-Aldrich) while shaking gently. Cells were then washed using distilled water to remove free dye. In addition, free dye was eluted from these cells using 100% isopropanol, after which absorbance at 490 nm was assessed via spectrophotometry [31] .
qRT-PCR TRIzol (Invitrogen) was used to extract cellular RNA as above, and a cDNA Reverse Transcription Kit (Thermo, CA, USA) was then utilized for cDNA preparation. All qRT-PCR reactions were conducted using a SYBR Premix Ex Taq kit (Toyobo, Osaka, Japan) with an ABI Prism 7500 instrument (Applied Biosystems). The primers were used in this study are compiled in Table S1 (Supplementary Files).
Relative gene expression was quanti ed using the 2 -ΔΔCt method [32] [33] .