Expression profiling of long noncoding RNAs in neonatal and adult mouse testis

In recent years, advancements in genome-wide analyses of the mammalian transcriptome have revealed that long noncoding RNAs (lncRNAs) is pervasively transcribed in the genome and an increasing number of studies have demonstrated lncRNAs as a new class of regulatory molecules are involved in mammalian development (Carninci et al. (2005); Fatica and Bozzoni (2014)), but very few studies have been conducted on the potential roles of lncRNAs in mammalian testis development. To get insights into the expression patterns of lncRNA during mouse testis development, we investigated the lncRNAs expression profiles of neonatal and adult mouse testes using microarray platform and related results have been published (Sun et al., PLoS One 8 (2013) e75750.). Here, we describe in detail the experimental system, methods and validation for the generation of the microarray data associated with our recent publication (Sun et al., PLoS One 8 (2013) e75750.). Data have been deposited to the Gene Expression Omnibus (GEO) database repository with the dataset identifier GSE43442.


a b s t r a c t
In recent years, advancements in genome-wide analyses of the mammalian transcriptome have revealed that long noncoding RNAs (lncRNAs) is pervasively transcribed in the genome and an increasing number of studies have demonstrated lncRNAs as a new class of regulatory molecules are involved in mammalian development (Carninci et al. (2005); Fatica and Bozzoni (2014)), but very few studies have been conducted on the potential roles of lncRNAs in mammalian testis development. To get insights into the expression patterns of lncRNA during mouse testis development, we investigated the lncRNAs expression profiles of neonatal and adult mouse testes using microarray platform and related results have been published (Sun et al., PLoS One 8 (2013) e75750.). Here, we describe in detail the experimental system, methods and validation for the generation of the microarray data associated with our recent publication (Sun et  3. Experimental design, materials and methods

Collection of testis samples
Twenty-one neonatal (6-day-old) and six adult (8-week-old) male C57BL/6 mice were purchased from SLAC Laboratory Animal Co., Shanghai, China. Mice in each age group were divided into three groups to provide three biological replicates for microarray analysis. Mice were sacrificed by cervical dislocation. Whole testes were surgically removed from mice and were immediately snap-frozen in liquid nitrogen and ground to a fine powder with mortar and pestle that was pre-cooled with liquid nitrogen and then Trizol reagent (Invitrogen) was added to continue grinding.

RNA extraction
Total RNA was extracted according to manufacturers instructions of Trizol reagent. RNA quantity and quality were measured by NanoDrop ND-1000 spectrophotometer (Thermo Scientific). RNA integrity and genomic DNA contamination were assessed by denaturing agarose gel electrophoresis and Bioanalyzer 2100 (Agilent Technologies).

RNA labeling and array hybridization
Sample labeling and array hybridization were performed according to the Agilent One-Color microarray-based gene expression analysis protocol (Agilent Technology). Briefly, for RNA labeling, 1 μg of total from each sample was labeled with Cy3-dCTP using RNA Spike-In Kit (Agilent, 5188-5282) and Quick Amp labeling kit (Agilent, 5190-0442), the labeled cRNAs were purified by RNAeasy Mini Kit (Qiagen), the concentration and specific activity of the labeled cRNAs (pmol Cy3/μg cRNA) were measured by NanoDrop ND-1000 Spectrophotometer (Thermo Scientific). For array hybridization, 1 μg of each labeled cRNA was fragmented by adding 11 μl 10Â Blocking Agent and 2.2 μl of 25 Â Fragmentation Buffer (Agilent), then heated the mixture at 60 1C for 30 min, finally 55 μl 2 Â GE Hybridization buffer (Agilent) was added to dilute the labeled cRNA. 100 μl of hybridization solution was dispensed into the gasket slide and assembled to the lncRNA expression microarray slide (Mouse Stringent LncRNA microarray, 4 Â 44 K, ArrayStar). The slides were incubated for 17 h at 65 1C in Agilent's SureHyb Hybridization Chambers. After hybridization, microarrays were washed 1 min at room temperature with GE Wash Buffer 1 (Agilent) and 1 min with Wash buffer 2 (Agilent) at 37 1C, then dried immediately by brief centrifugation. The hybridized arrays were washed, fixed and scanned with using the Agilent DNA Microarray Scanner (part number G2505B).

Data analysis
Agilent Feature Extraction software (version 10.5.1.1) was used to extract raw data from scanned array images. Median normalization and subsequent data processing were performed with using the GeneSpring GX v11.0 software package (Agilent Technologies). After median normalization of the raw data and low intensity filtering, lncRNAs and mRNAs that at least 1 out of 6 samples have flags in Present ("All Targets Value") were chosen for further data analysis. The quality of lncRNA and mRNA was assessed by Box-Plot and Scatter-Plot. The Box Plot is commonly used for comparing the distributions of the intensities from all samples. After normalization, the distributions of log2-ratios among all tested samples are nearly the same. The Scatter-Plot is a visualization method used for assessing expression variation (or reproducibility) between the two compared arrays. Differentially expressed lncRNAs and mRNAs were screened through performing a Volcano plot filtering between the two samples. The threshold is Fold Change 4 ¼5.0 and p-value o ¼0.05 (Student's t-test). Hierarchical Clustering was performed using the Agilent GeneSpring GX software (version 11.0) (Fig. 1). GO (Gene Ontology) analysis and Pathway analysis was performed using Database for Annotation, Visualization and Integrated Discovery (DAVID) [4]. Raw and processed microarray data have been deposited to the Gene Expression Omnibus (GEO) database in the National Center for Biotechnology Information (NCBI), and can be accessed by the GEO accession number, GSE43442.

Validation of microarray data
To validate the microarray data, we investigated the expression level of eight differentially expressed lncRNAs in neonatal (6-day-old) and adult (8-week-old) mouse testis using quantitative real-time PCR (qRT-PCR). The results clearly showed that there was a excellent correlation (Spearman coefficient rho¼0.952, po0.01, n ¼8) between the microarray data and the qRT-PCR data ( Table 1), indicating that the microarray results were reliable. The primers used for qRT-PCR see Table 2. In addition, GO analysis revealed that up-regulated mRNAs in adult testis was significantly enriched in reproduction-related GO terms, such as sexual reproduction, multicellular organism reproduction, gamete generation and spermatogenesis and so on (shown in Fig. 2), indirectly indicating the microarray results were reliable.

Discussion
Genome-wide analyses of the mammalian transcriptome revealed that lncRNAs is pervasively transcribed in the genome and accumulating studies have demonstrated that lncRNAs as novel regulatory molecule plays critical roles in mammalian development [1,2], but little is known about the   AK011865  CCACTTCAAATGGGGAGGGT  GTGATTTGGGACACTGGAGAGAC  182  AK006530  CAAGTGCCTGGGATTGTGGATAC  CAGCCCACTGTCCAGGTCATCTC  211  AK053216  GCCCAAGCGGTCACTCAGTATCA  AGTGGTTCAAGTTACTGCCGCTG  287  AK018942  GCCTTTCCCAAATGTCTGTTCCT  TCAGGTTCAGGGAGTGCTTCTTT  245  AK006015  GCCACACCACCATCCACTATT  GGAGGCGAAACCAGGTAGATT  161  AK004447  TGTAGCATCAAACATTCACGGA  GACAGAAGGACACAGGGCAAAC  196  AK033245  CAAACCCGCAGTTCTTCTCCTT  AGTGCCAGCCCGAGTTGTTC  205  AK140218  TTTACAAAAGGGAAAACTGAGGC  TAGGCTGATAAAGGGCTCAAAGTT  196  GAPDH  GTCGTGGAGTCTACTGGTGTC  GAGCCCTTCCACAATGCCAAA  240 potential roles of lncRNAs in mammalian testis development. Here, we described a dataset composed of lncRNA and mRNA expression profiling of neonatal (6-day-old) and adult (8-week-old) mouse testis. With this experiment, we were able to show 3025 of lncRNAs and 5964 of mRNA are differentially expressed between neonatal and adult mouse testes. Some known haploid male germ cell-specific lncRNAs was also found differentially expressed in this study, for example, Aldoart2, Speer5-ps1 and Speer9-ps1 [5,6]. The dynamic change of lncRNA expression during mouse testis postnatal development indicated that lncRNAs might play crucial roles in mammalian testis development and spermatogenesis. Thus, this experiment provides a solid foundation for the identification and characterization of key lncRNAs involved in testis development or spermatogenesis.