Computational protocol: MicroRNA-378 limits activation of hepatic stellate cells and liver fibrosis by suppressing Gli3 expression

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Protocol publication

[…] Total RNA was extracted from liver tissues or cells by using TRIzol reagent (Ambion, Life Technologies) or miRNeasy Mini Kit (Qiagen, Valencia, CA, USA) to enrich miRNA quantity. The concentration and purity of RNA were determined using a nanodrop. Template complementary DNA was synthesized from total RNA using the SuperScript First-strand Synthesis System (Invitrogen, Life Technologies) or miScript Reverse Transcriptase Kit (Qiagen) according to the manufacturer's protocols. We performed the real-time qRT–PCR analysis by using Power SYBR Green Master Mix (Applied Biosystem, Life technologies) or miScript SYBR Green PCR Kit (Qiagen) on the manufacturer's specifications (Eppendorf, Mastercycler Real-Time PCR). All reactions were triplicated and data were analysed according to the ΔΔCt method. 40S ribosomal protein S9 mRNA and 18S ribosomal RNA for mRNA and U1A small nuclear RNA (RNU1A) for miRNA were used for normalization of the expression level. In data analysis for the expression of miR-378a-3p and gli3 in human samples, average of expression of miR-378a-3p or gli3 from all paired non-tumour and tumour tissues was calculated. Next, individual value of expression relative to the mean value was presented as scatter plot and mean±s.e.m. of each group was graphed. The sequences of all primers used in this study are summarized in . All PCR products were directly sequenced for genetic confirmation (Macrogen). [...] Results are expressed as the mean±s.e.m. Statistical significances between control and treated groups or subgroups were analysed by the unpaired two-sample Student's t-test or one-way analysis of variance followed by a post-hoc Tukey's test. Data for in vivo effect of miR-378 in the liver were first analysed with the non-parametric Kruskal–Wallis test and then the differences between subgroups were further analysed by the two-sample Student's t-test. Differences were considered as significant when P-values are <0.05. Results from HCC patients were expressed as individual values of relative gene expression and the mean±s.e.m., to show distributions of most variables in patients. Difference between non-tumour and tumour group was determined using the paired two-sample Student's t-test and considered as significant when P-values are <0.05. The degree of correlation between the expression levels of miR-378a-3p and gli3 in liver tissues of HCC and between the level of miR-378a-3p and hydroxyproline content or Gli3 in liver tissues of CCl4-treated mice were analysed by the Spearman's rank correlation coefficient (ρ). Statistical analyses were performed using IBM SPSS Statistics 21 software (Release version, IBM Corp., Armonk, NY, USA). […]

Pipeline specifications

Software tools ddCt, SPSS
Applications Miscellaneous, qPCR
Organisms Mus musculus, Zea mays subsp. mays, Homo sapiens
Diseases Carcinoma, Hepatocellular, Neoplasms
Chemicals Carbon Tetrachloride