The effects of leptin-A morpholino oligonucleotides and recombinant leptin-A on embryonic zebrafish transcriptome content were evaluated using Affymetrix 1.1 ST gene array strips.

Microarray transcriptional analysis of leptin signaling knockdown and rescue in 72-hour post fertilization zebrafish

Leptin-A mediates central endocrine and phosphatidylinositol signaling pathways in 72-hour embryonic zebrafish.

Affymetrix GeneChip Zebrafish 1.1 ST Array [ZebGene-1_1-st]

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The effects of leptin-A morpholino oligonucleotides and recombinant leptin-A on embryonic zebrafish transcriptome content were evaluated using Affymetrix 1.1 ST gene array strips.


Fertilized zebrafish embryos were serially collected, cleaned, and segregated from adults after spawning (< 0.25 hours post fertilization).  After microinjection, zebrafish developed to 72 hours post fertilization. For each biological replicate, a total of 5 embryos were homogenized for microarray processing.

Total RNA was isolated from 72-hour zebrafish embryos with TRIzol reagent (Invitrogen; Carlsbad, CA; 15596026).  RNA was DNase-treated with the Turbo-DNA-free kit (Ambion; Foster City, CA; AM1907), then washed with the RNeasy Minelute cleanup kit (Qiagen; Hilden Germany; 74204).  RNA integrity (RIN) and quantity were scored with an Agilent 2100 electrophoretic bioanalyzer (RIN ≥ 9.0; Agilent; Santa Clara, CA).  RNA was immediately shipped on dry ice to The University of Michigan’s Microarray Core Facility (Ann Arbor, Michigan, USA) for library synthesis and microarray scanning.

A total of 16 biotinylated sense-strand cDNA libraries were prepared for transcriptomic analysis.  cDNA was generated from 250 ng of quality-assured total RNA with the WT Expression kit (Ambion; Foster City, CA; 4411973). cDNA libraries were biotinylated using the GeneChip Terminal Labeling kit (Ambion; Foster City, CA; 901525).

Following fragmentation, 5.5 ug of biotinylated cDNA was hybridized to Affymetrix Zebrafish 1.1 ST gene array strips (Affymetrix; Santa Clara, CA; 901802) for 20 hours at 48oC.

2 nL of 0.4 mM leptin-A antisense morpholino oligonucleotides (5’-TTG AGC GGA GAG CTG GAA AAC GCA T -3’) (Gene Tools; Philomath, OR), reconstituted in Daneau buffer ([58 mM NaCl, 0.7 mM KCl, 0.4 mM MgSO4, 0.6 mM Ca(NO3)2, 5.0 mM HEPES pH 7.6]), were microinjected into 1 - 2 cell stage zebrafish embryos.  A second group of leptin-A rescue microinjections were prepared with 30 µM recombinant zebrafish leptin-A protein stock solution in 50 mM Tris, pH 8.0 (> 90% pure, GenScript; Piscataway, NJ) mixed 1:1 with 0.4 mM leptin-A morpholinos.  Age-matched uninjected control embryos served as a baseline comparison to each treatment.

Microarray scanning was performed by The University of Michigan Core Facility (Ann Arbor, Michigan, USA) on the Affymetrix GeneAtlas system v1.0.4.267 (Affymetrix; Santa Clara, CA).

Microarray .CEL files were processed with the Robust Multichip Average method in “R” statistical environment with the ‘oligo’ library package using core summarization.  Affymetrix probeset annotations were derived from the ‘pd.zebgene.1.1.st’ library package.  Assessments of differential expression were performed with the linear models for microarray data (limma) library package which includes an empirical Bayesian method that shrinks sample variances towards pooled estimates of variance (Smyth 2004).  Differentially expressed genes were called at p.value < 0.01 and log2 fold change > 0.5 or < -0.5.  P.values were adjusted for multiple testing using the method of Benjamini and Hochberg.  Differentially expressed genes were evaluated for three pairwise comparisons: (I.) leptin-A morphant compared to control, (II.) leptin-A rescue compared to leptin-A morphant,(III.) leptin-A rescue compared to control.

Microarray transcriptional analysis of leptin signaling knockdown and rescue in 72-hour post fertilization zebrafish

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