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Summary
Purpose: The study was designed to determine expression differences in brain regions of F344 and HIV-1 Transgenic rats by using RNA-sq analysis. Methods: 144 RNA samples (2 strains, 2 treatments, 3 regions, and 12 animals per group) were analyzed. Following deep-sequencing analysis of 50-bp paired-end reads of RNA-Seq, we used Bowtie/Tophat/Cufflinks suites to align these reads into transcripts based on the Rn4 rat reference genome and to measure the relative abundance of each transcript. MATLAB was used to conduct all statistical analysis. qRT–PCR validation was performed using TaqMan and SYBR Green assays fo soem representative genes. Results: Statistical and bioinformatic analyses on each brain region in the two strains revealed that immune response- and neurotransmission-related pathways were altered in the HIV-1Tg rats, with brain region differences. Other neuronal survival-related pathways, including those encoding myelin proteins, growth factors, and translation regulators, were altered in the HIV-1Tg rats in a brain region-dependent manner. After nicotine expousure, 20% of the altered genes in the HIV-1Tg rat were affected by nicotine in each brain region, with the expression of most restored. Analysis of the restored genes showed distinct pathways corrected by nicotine in different brain regions of HIV-1Tg rats.
Conclusions: The abnormal gene expression pattern discovered in HIV-1Tg rats suggest mechanisms underlying the deficits in learning and memory and vulnerability to drug addiction and other psychiatric disorders observed in HIV positive patients. The gene expression pattern in the HIV-1Tg rats after nicotine exposure indicate that cholinergic modulators such as nicotine may have beneficial effects on HIV-1-induced neurologic deficits.
Experiment design
144 RNA samples (2 strains, 2 treatments, 3 regions, and 12 animals per group) were analyzed.
About cases
The Animal Care and Use Committee of both the Seton Hall University and University of Virginia approved this study. Adult male HIV-1Tg rats and F344 background control rats (n = 12 per group) were purchased from Harlan Inc. (Indianapolis, IN). All rats were double housed in standard plastic cages and maintained in a temperature-controlled environment with a 12 h light/dark cycle and fed a standard rat diet and water ad libitum. The animals were monitored daily, and their cage bedding was changed twice a week. All animals were participants in a previously reported behavioral study [31]. All experimental procedures were conducted during the light cycle in accordance with the Animal Care and Use Committees of both participating institutions.
About tissue
Using a rat brain matrix, slices of approximately 1 mm were taken from each brain, and the slices that contained the PFC, HIP, and dorsal STR were identified according to a rat brain atlas [35]. Tissues from specific regions of interest were collected bilaterally from each brain using a 3.00-mm Harris Micro-Punch (GE Healthcare Life Sciences, Piscataway, NJ, USA) and stored at −80°C until use.
About platform
GPL14844Illumina HiSeq 2000 (Rattus norvegicus)
About data processing
The extraction of 50-bp length paired-end reads was achieved using CASAVA (Illumina Pipeline v1.38). For each sample, reads with a quality score of ≥Q30 that passed filtering were used to generate a complete FASTQ file, which was then mapped to UCSC Rat reference [build Rn4] (ftp://ftp.cbcb.umd.edu/pub/data/bowtie_indexes/rn4.ebwt.zip) using TopHat with the default parameter setting of 40 alignments per read and up to 2 mismatches per alignment. The sequence alignment files (BAM) were analyzed using RSeQC package [36] for quality control analysis, which includes the mRNA fragment insert size, base quality distribution, reads mapping distribution, and splicing distribution analysis.
The resulting aligned reads were then analyzed with Cufflinks suite (http://cufflinks.cbcb.umd.edu) [37], which assembles the aligned reads into transcripts and measures their relative abundance. The expression of each transcript was quantified as the number of reads mapping to a gene divided by the gene length in kilobases and the total number of mapped reads in millions, which is called fragments per kilobase of exon per million fragments mapped (FPKM). All the junctions identified by Cufflink were compared on the basis of the junction and splicing site provided by reference transcript annotation GTF files to identify known and novel junctions. Then, Cuffcompare merged all the transcripts from different samples to a final transcript annotation GTF file, reported changes in the relative abundance of transcripts sharing a common transcription start site, and indicated the relative abundance of the primary transcripts of each gene crossing all the samples.
Contributors
Conceived and designed the experiments: MDL SLC. Performed the experiments: JC SW SS MV. Analyzed the data: JC SW JW JZM. Wrote the paper: MDL JC SW JW JZM SLC.
Li MD, Cao J, Wang S, Wang J, Sarkar S, Vigorito M, Ma JZ, Chang SL
Citation
- Li MD, Cao J, Wang S, Wang J et al. Transcriptome sequencing of gene expression in the brain of the HIV-1 transgenic rat. PLoS One2013;8(3):e59582. PMID: 23536882
- Cao J, Wang S, Wang J, Cui W et al. RNA deep sequencing analysis reveals that nicotine restores impaired gene expression by viral proteins in the brains of HIV-1 transgenic rats. PLoS One 2013;8(7):e68517. PMID: 23874651
Acknowledgment
We thank Drs. David L. Bronson and Louaine L. Spriggs for their excellent editing of this manuscript.