dna microarray analysis total rnas  (Thermo Fisher)

Journal: Molecular cancer research : MCR

Article Title: TFE3 Xp11.2 translocation renal cell carcinoma mouse model reveals novel therapeutic targets and identifies GPNMB as a diagnostic marker for human disease

doi: 10.1158/1541-7786.MCR-18-1235

Figure Lengend Snippet: (a) Numbers of genes differentially expressed between PRCC-TFE3;KSP-Cre+ kidneys and PRCC-TFE3;KSP-Cre- kidneys. (b, c) Volcano plot of gene expression changes for 4 month-old (b) and 7 month-old (c) PRCC-TFE3;KSP-Cre- kidneys and PRCC-TFE3;KSP-Cre+ kidneys. The x-axis specifies the fold-changes [Log2(Cre(+)/Cre(-))] and the y-axis specifies the negative log to the base 10 of the t-test q-values. Red and blue dots represent genes expressed at significantly higher or lower levels (>2 fold vs. <-2 fold) in PRCC-TFE3;KSP-Cre+ kidneys (q<0.05). Gpnmb was one of the most significant genes showing increased expression in both 4 month-old and 7 month-old PRCC-TFE3;KSP-Cre+ kidneys. Ret was identified as one of the highly expressed and druggable receptor tyrosine kinases in both 4 month-old and 7 month-old PRCC-TFE3;KSP-Cre+ kidneys. (d) Expression of Gpnmb was quantified by qRT-PCR analysis of 8 month-old PRCC-TFE3;KSP-Cre- mouse kidneys (n=6) and PRCC-TFE3;KSP-Cre+ mouse kidneys (n=7). Data are represented as box-and-whisker plot. (unpaired t-test) (e, f) Gene Set Enrichment Analysis (GSEA) of microarray data from 4 month old PRCC-TFE3; KSP-Cre+ mouse kidneys (n=4) vs 4 month old PRCC-TFE3; KSP-Cre- mouse kidneys (n=4) (e) and 7 month old PRCC-TFE3; KSP-Cre+ mouse kidneys (n=4) vs 7 month old PRCC-TFE3; KSP-Cre- mouse kidneys (n=3) (f). Significant enrichment of genes associated with EGFR activation (e) and RET activation (f) was seen in kidneys from PRCC-TFE3; KSP-Cre+ mice. NES: normalized enrichment score, NOM p-Value: Nominal p value. (g) Expression of Ret was quantified by qRT-PCR analysis of 8 month-old PRCC-TFE3;KSP-Cre- mouse kidneys (n=6) and PRCC-TFE3;KSP-Cre+ mouse kidneys (n=7). Data are represented as box-and-whisker plot. (unpaired t-test) (h) H&E staining of 7 month-old PRCC-TFE3;KSP-Cre- and (i) PRCC-TFE3;KSP-Cre+ mouse kidneys. (j-o) Representative immunostaining for TFE3 (j, k), Gpnmb (l, m), and Ret (n, o) on serial sections of 7 month-old PRCC-TFE3;KSP-Cre- (j, l, n) and PRCC-TFE3;KSP-Cre+ (k, m, o) kidneys. Lower panels are higher magnified images of the rectangular areas in upper panels. (p-v) Treatment of PRCC-TFE3;KSP-Cre+ mice with vandetanib, an inhibitor of RET. Representative coronal T2 weighted MRI images of vehicle treated (p, q) and vandetanib treated (100mg/kg) (r, s) PRCC-TFE3;KSP-Cre+ mice chronologically taken on day 0 and day 116 of the study. Red arrows indicate the tumors which were tracked for growth. (t, u) Representative tumor growth curves of vehicle treated (t) and vandetanib treated (u) PRCC-TFE3;KSP-Cre+ mice shown in (p, q) and (r, s), respectively. The largest dimension of each tumor was measured from sequential MRI images taken on day 0, 30, 60, 95, and 116 of treatment. (v) Nonlinear regression analysis of % tumor growth in the vehicle treated group (black dots) and vandetanib treated group (red square). (****p<0.0001) (w) Representative immunostaining for RET on human TFE3-RCC demonstrates significant cytoplasmic staining.

Article Snippet: DNA Microarray Analysis Total RNA was isolated from flash frozen mouse kidneys using TRIzol reagent (Invitrogen). cDNA preparation and hybridization of the probe arrays were performed according to the manufacturer’s instructions (Affymetrix).

Techniques: Gene Expression, Expressing, Quantitative RT-PCR, Whisker Assay, Microarray, Activation Assay, Staining, Immunostaining