Journal: PLoS ONE

Article Title: MicroRNA-376a Regulates 78-Kilodalton Glucose-Regulated Protein Expression in Rat Granulosa Cells

doi: 10.1371/journal.pone.0108997

Figure Lengend Snippet: List of differentially expressed (upregulated) miRNAs in rat ovaries following treatment with hCG for 6 h.

Article Snippet: The total RNA was then reverse transcribed using specific RT primers from a TaqMan MicroRNA Assay Kit (rno-miR-144: 197375, rno-miR-376a: 001069, rno-miR-451: 001141, and 4.5S RNA(H): 001716 as an internal control; Ambion Inc.).

Techniques:

Journal: PLoS ONE

Article Title: MicroRNA-376a Regulates 78-Kilodalton Glucose-Regulated Protein Expression in Rat Granulosa Cells

doi: 10.1371/journal.pone.0108997

Figure Lengend Snippet: Female 21-day-old rats injected subcutaneously with PMSG (30 IU/rat), followed by hCG (20 IU/rat) 48 h later, were sacrificed at the indicated times. The ovaries were removed, and total RNA was isolated. Rat GRP78 mRNA (A), rno-miR-144 (B), rno-miR-376a (C), and rno-miR-451 (D) expression levels were measured using real-time RT-PCR as described in the . The amount of rat GRP78 mRNA, rno-miR-144, rno-miR-376a, and rno-miR-451 in the hCG 0 h group was set at 1. Data were normalized to 18S rRNA (for GRP78 mRNA) and 4.5S RNA(H) (for rno-miR-144, rno-miR-376a, and rno-miR451) levels in each sample and represent the mean ±SE of three independent experiments. *, significantly different from the control value at hCG 0 h, P <0.05.

Article Snippet: The total RNA was then reverse transcribed using specific RT primers from a TaqMan MicroRNA Assay Kit (rno-miR-144: 197375, rno-miR-376a: 001069, rno-miR-451: 001141, and 4.5S RNA(H): 001716 as an internal control; Ambion Inc.).

Techniques: Injection, Isolation, Expressing, Quantitative RT-PCR

Journal: PLoS ONE

Article Title: MicroRNA-376a Regulates 78-Kilodalton Glucose-Regulated Protein Expression in Rat Granulosa Cells

doi: 10.1371/journal.pone.0108997

Figure Lengend Snippet: Primary rat granulosa cells were prepared, and the indicated reagents were added to the medium after 24 h of culture. Cells were then incubated with FSH (30 ng/mL) and estradiol (10 nM) for 48 h. Subsequently, hCG (30 ng/mL) was added to the culture medium, as described in the . Total RNA was isolated, and GRP78 mRNA (A), rno-miR-144 (B), rno-miR-376a (C), and rno-miR-451 (D) expression levels were measured using real-time RT-PCR as described in the . The amounts of GRP78 mRNA, rno-miR-144, rno-miR-376a, and rno-miR-451 in the hCG 0 h group were set at 1. Data were normalized for 18S rRNA (for GRP78 mRNA) and 4.5S RNA(H) (for rno-miR-144, rno-miR-376a, and rno-miR-451) levels in each sample and represent the mean ±SE of 3 independent experiments. *, significantly different from the control value at hCG 0 h, P <0.05.

Article Snippet: The total RNA was then reverse transcribed using specific RT primers from a TaqMan MicroRNA Assay Kit (rno-miR-144: 197375, rno-miR-376a: 001069, rno-miR-451: 001141, and 4.5S RNA(H): 001716 as an internal control; Ambion Inc.).

Techniques: Incubation, Isolation, Expressing, Quantitative RT-PCR

Journal: PLoS ONE

Article Title: MicroRNA-376a Regulates 78-Kilodalton Glucose-Regulated Protein Expression in Rat Granulosa Cells

doi: 10.1371/journal.pone.0108997

Figure Lengend Snippet: Primary rat granulosa cells were prepared, and the indicated reagents were added to the medium after 24 h of culture. Cells were then incubated with FSH (30 ng/mL) and estradiol (10 nM) for 48 h in the same way as described in Fig. 2. The time after 48 h of incubation with FSH and estradiol was considered “0 h.” Total RNA was isolated, and rno-miR-376a expression levels was determined using real-time RT-PCR at the indicated time. The amounts of rno-miR-376a in the 0 h group were set at 1. Data were normalized for 4.5S RNA(H) levels in each sample and represent the mean ±SE of 3 independent experiments.

Article Snippet: The total RNA was then reverse transcribed using specific RT primers from a TaqMan MicroRNA Assay Kit (rno-miR-144: 197375, rno-miR-376a: 001069, rno-miR-451: 001141, and 4.5S RNA(H): 001716 as an internal control; Ambion Inc.).

Techniques: Incubation, Isolation, Expressing, Quantitative RT-PCR

Journal: PLoS ONE

Article Title: MicroRNA-376a Regulates 78-Kilodalton Glucose-Regulated Protein Expression in Rat Granulosa Cells

doi: 10.1371/journal.pone.0108997

Figure Lengend Snippet: Primary rat granulosa cells were prepared, and the indicated reagents were added to the medium after 24 h of culture. The cells were incubated with FSH (30 ng/mL) and estradiol (10 nM) for 48 h. Pre-miR-376a (precursor) or Anti-miR-376a (inhibitor) was transfected into the cells, and 30 ng/mL hCG was added 12 h later. The effects of precursor and inhibitor on the expression of GRP78 mRNA were measured using real-time RT-PCR, as described in the . The expression of GRP78 mRNA at hCG 0 h in the control was set at 1. Each value represents the mean ±SE of three independent experiments.

Article Snippet: The total RNA was then reverse transcribed using specific RT primers from a TaqMan MicroRNA Assay Kit (rno-miR-144: 197375, rno-miR-376a: 001069, rno-miR-451: 001141, and 4.5S RNA(H): 001716 as an internal control; Ambion Inc.).

Techniques: Incubation, Transfection, Expressing, Quantitative RT-PCR

Journal: PLoS ONE

Article Title: MicroRNA-376a Regulates 78-Kilodalton Glucose-Regulated Protein Expression in Rat Granulosa Cells

doi: 10.1371/journal.pone.0108997

Figure Lengend Snippet: (A) Arrangement of rno-miR-376a and GRP78 mRNA and a schematic drawing of the predicted rno-miR-376a-binding site in the 3′-UTR of GRP78 mRNA. (B) Schematic drawings of the pMIR-REPORT luciferase vectors used in our experiment. To identify the rno-miR-376a-binding site in the 3′-UTR of GRP78 mRNA, luciferase reporter vectors were generated as described in the . (C) Luciferase activity was measured to identify the rno-miR-376a-binding site in the 3′-UTR of GRP78 mRNA. HEK293 cells were prepared, and the cells were transfected with 200 ng of each reporter vector with 50 nM Pre-miR-376a (precursor) or Anti-miR-376a (inhibitor) as described in the . For transfection normalization, the cells were also transfected with the pMIR-REPORT βgal vector. Luciferase activity was measured 24 h after transfection. The activity of the control (empty vector) was set at 1. Each value represents the mean ±SE of three independent experiments. *, significantly different from the control value, P <0.05.

Article Snippet: The total RNA was then reverse transcribed using specific RT primers from a TaqMan MicroRNA Assay Kit (rno-miR-144: 197375, rno-miR-376a: 001069, rno-miR-451: 001141, and 4.5S RNA(H): 001716 as an internal control; Ambion Inc.).

Techniques: Binding Assay, Luciferase, Generated, Activity Assay, Transfection, Plasmid Preparation