Tissue collection and RNA isolation
Bovine tissue samples including adult liver, lung, thymus, kidney, muscle, heart, spleen, cortex (brain), pituitary, adrenal, testis, ovary, and fetal testis and ovaries, were collected at a local slaughterhouse. All samples were frozen in liquid nitrogen and stored at -80°C until RNA isolation. Total RNA was isolated from these tissues using TRIzol reagent (Invitrogen, Carlsbad, CA) and treated with DNase (Promega, Madison, WI) according to manufacturer's protocols.
RT-PCR analysis of bovine NPM2 mRNA expression
Total RNA from various bovine tissues was used to generate cDNA using oligo (dT)18 primer and Superscript III reverse transcriptase (Invitrogen, Carlsbad, CA). Negative control reactions (without the enzyme) were carried out to confirm the absence of genomic DNA contamination. First-strand cDNA was used as a template for PCR amplification of a 329 bp fragment using bovine NPM2 gene-specific primers (Additional file 1, Table S1). The PCR was performed using 35 cycles of 94°C for 30 sec, 59°C for 45 sec and 72°C for 30 sec, and a final extension at 72°C for 10 min. Bovine ribosomal protein L19 (RPL19) gene was used as a positive control.
Cloning of bovine NPM2 cDNA by PCR and RACE
PCR primers were designed based on conserved regions of human (NM_182795) and mouse (NM_181345) NPM2 sequences to amplify a 329 bp cDNA fragment from a fetal ovary sample (230 days of gestation). The product was cloned using TOPO® TA cloning kit (Invitrogen, Carlsbad, CA) and sequenced. Primers for 5' and 3'RACE were designed based on the obtained bovine NPM2 cDNA sequence (Additional file 1, Table S1). RACE experiments were performed to obtain the 5' and 3' ends of bovine NPM2 cDNA using the second generation 5'/3'RACE kit (Roche Diagnostics, Indianapolis, IN) following the manufacturer's protocol. Total RNA from bovine fetal ovary was used to generate cDNA with either a gene-specific primer (5'RACE) or an oligo d(T)-anchor primer (3'RACE) followed by nested PCR using gene-specific primers in conjunction with d(T) anchored primers provided in the kit. The specific PCR products were cloned using the TOPO® TA cloning kit (Invitrogen, Carlsbad, CA). The RACE products were sequenced at the University of Illinois Core DNA Sequencing Facility (Urbana, IL).
Quantitative real time PCR analysis
Expression of bovine NPM2 mRNA during oocyte maturation and early embryonic development was determined by real time PCR as described previously  using primers listed in Additional file 1, Table S1. GV and MII stage oocytes, pronuclear, 2-cell, 4-cell, 8-cell, 16-cell, morula and blastocyst stage embryos (n = 5 pools of 10 each) were obtained by in vitro fertilization of abattoir derived oocytes as described . Total RNA from oocytes and embryos was isolated using the RNAqueous®-Micro kit (Ambion Inc., Austin, TX). Spiked green fluorescent protein (GFP) synthetic RNA was used as an exogenous control to account for variations in RNA recovery and efficiency of cDNA synthesis between samples.
Quantitative real time PCR analysis of bovine NPM2 mRNA expression in oocytes from growing and persistent dominant follicles was performed as described previously . Oocytes from growing (day 6) and persistent follicles (day 13, estrus = day 0) were used in this analysis. Total RNA isolated from individual oocytes was subjected to linear amplification before real time PCR assay. Bovine HIST2H2AA4 gene (BF076713) was used as an endogenous control for data normalization as expression of this gene does not differ in oocytes from the two types of follicles . Primers for this gene are listed in Additional file 1, Table S1.
Quantitative real time PCR analysis of miR-181a expression in oocytes and early stage embryos was performed as described . miR-125b was used as an endogenous control to normalize the target miRNA because this miRNA is expressed consistently in preimplantation mouse embryos .
Western blot analysis
Western blot analysis of bovine NPM2 protein expression in oocytes and early embryos was performed as previously described . The oocyte and embryo samples (50 oocytes/embryos each lane) were purchased from Bomed Inc. (Madison, WI). The primary antibody (anti-bovine NPM2) was prepared commercially by GenScript Corporation (Piscataway, NJ). It was generated by immunizing rabbits with a 15-amino acid synthetic peptide (ERPTWTFKPQKVGKC, amino acid position 26-39) of bovine NPM2 protein. Unpurified antiserum from the third bleed was used in the study.
Preparation of expression constructs
The plasmid expressing bovine NPM2 was constructed by cloning of the full length bovine NPM2 cDNA into pcDNA3.1 expression vector (Invitrogen, Carlsbad, CA). PCR primers (Additional file 1, Table S1) containing BamHI (forward) and XhoI (reverse) restriction sites were designed to amplify the full length bovine NPM2 cDNA using a fetal ovary cDNA sample (230 days of gestation). The amplified PCR product was cloned using TOPO® TA cloning kit (Invitrogen, Carlsbad, CA) followed by subcloning into the BamHI and XhoI sites of pcDNA3.1. The plasmid designed to express the bovine miR-181a was prepared by cloning a 262 bp genomic fragment containing the pre-miR-181a into pcDNA3.1. Primers containing BamHI (forward) and PmeI (reverse) restriction sites (Additional file 1, Table S1) were used to amplify the 262 bp DNA fragment from kidney genomic DNA. Following TA cloning the product was cloned into the BamHI and PmeI sites of pcDNA3.1. Both constructs were sequenced to ensure that no mutations were introduced during PCR amplification.
Cell culture and transfection experiments
HeLa cells from ATCC (Manassas, VA) were grown at 37°C in a humidified incubator containing 5% CO2 in Dulbecco's modified Eagle's medium (DMEM; Invitrogen, Carlsbad, CA) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin. Stable HeLa cells expressing bovine miR-181a was generated by transfecting the cells with miRNA-181a plasmid followed by selection in G418. Transfection experiments (n = 5) were conducted using 6-well culture dishes. HeLa cells were passed at least 12 hours prior to all transfection experiments. HeLa cells expressing bovine miR-181a were transfected with NPM2 plasmid (1 μg) or pcDNA3.1 vector (1 μg) using FuGene® 6 transfection reagent (Roche Applied Science, Indianapolis, IN) according to the manufacturer's instructions. Control Hela cells not expressing bovine miR181a were also transfected with NPM2 plasmid or pcDNA3.1 vector. Twenty-four hours post-transfection, all cells were harvested by trypsinization. Cell pellets were lysed in cell lysis buffer (100 mM sodium β-glycerophosphate, 20 mM HEPES, 15 mM MgCl2, 5 mM EGTA, 100 mM 4-amidinophenylmethylsulfonyl fluoride, 3 mg/ml leupeptin, and 10 mg/ml aprotinin, pH 7.5) followed by sonication using an Ultrasonic Homogenizer (BioLogics, Manassas, VA). Western blot analysis of NPM2 protein expression in these cells was performed using anti-bovine NPM2 antibody as described above. Detection of GAPDH (protein loading control) was performed using anti-GAPDH antibody (Ambion, Austin, TX). Density of the protein bands was determined using a densitometer and NPM2 protein expression was normalized with GAPDH.
Differences in expression of NPM2 mRNA and protein and miRNA-181a between samples were analyzed by GLM procedures of SAS with LS means (SAS 9.1.3, SAS Institute Inc., Cary, NC). Different letters indicate significant differences (P < 0.05).