age: 32120 days old Sex: M tissue: prefrontal cortex of the brain batch: 1 individual no: 8
Treatment protocol
We used prefrontal cortex (PFC) and cerebellar cortex (CBC) samples from postmortem brains of human, chimpanzee, and rhesus macaque individuals of different age. Human samples were obtained from the NICHD Brain and Tissue Bank for Developmental Disorders at the University of Maryland, USA, the Netherlands Brain Bank, Amsterdam, Netherlands, and the Chinese Brain Bank Center, Wuhan, China. Informed consent for use of human tissues for research was obtained in writing from all donors or their next of kin. All subjects were defined as normal by forensic pathologists at the corresponding brain bank. All subjects suffered sudden death with no prolonged agonal state. Chimpanzee samples were obtained from the Yerkes Primate Center, GA, USA, the Anthropological Institute & Museum of the University of Zürich-Irchel, Switzerland, and the Biomedical Primate Research Centre, the Netherlands. Rhesus macaque samples were obtained from the Suzhou Experimental Animal Center, China. All non-human primates used in this study suffered sudden deaths for reasons other than their participation in this study and without any relation to the tissue used. CBC dissections were made from the cerebellar cortex. PFC dissections were made from the frontal part of the superior frontal gyrus. All samples contained an approximately 2:1 grey matter to white matter volume ratio.
Extracted molecule
total RNA
Extraction protocol
Total RNA was extracted from dissected CBC and PFC samples (humans, n=14; chimpanzees, n=11; macaques, n=8, respectively; Table S1), using the Ambion® mirVana miRNA isolation kit. Two individuals in human PFC and CBC sets and 5 individuals in chimpanzee PFC and CBC sets were measured in both batches to estimate the technical variance.
Label
Cy3
Label protocol
100 ng of dephosphorylated RNA was labeled with Cyanine 3-pCp following standard Agilent protocols.
Hybridization protocol
The hybridization mix was prepared using the Human miRNA Microarray kit (Agilent Technologies) following the manufacturer's instructions. Labeled miRNA from each sample was hybridized to one Agilent-021827 Human microRNA Microarray (v3, based on the Sanger miRBase Release 12.0). Hybridization was performed at 55C for 20h. Washing was performed using the Gene Expression Wash Buffer kit (Agilent Technologies) following the manufacturer's instructions. For human and chimpanzee, the microarray experiments were carried out in two batches. Batches were balanced with respect to samples' age.
Scan protocol
Arrays were scanned by a DNA microarray scanner (Agilent G2565BA).
Description
MicroRNA expression data from post-mortem PFC of a 32120 days old human individual
Data processing
The Agilent G4462AA Feature Extraction software (v10.7.3.1) was used for image analysis with default settings. In the original analysis we processed the data using the following steps (i) the data was log2 transformed and normalized, separately for each brain region dataset, (ii) we removed samples with low detection values (total detected miRNAs <200) and two samples that appeared as outliers in a principle components analysis (samples Ptr_CBC_525days_indv12_batch1, Hsa_CBC_1630days_indv5_batch2, Hsa_CBC_2922days_indv31_batch2, Hsa_CBC_8364days_indv32_batch2, Mml_CBC_3389days_indv25_batch1, Hsa_CBC_2days_indv1_batch2), (iii) we removed any batch effects by equalizing the mean expression level across batches, (iv) we calculated the mean profile per biological replicate, (v) we excluded any miRNAs that showed any sequence difference in their mature sequences among the 3 species. For this, we first extracted miRNA precursor sequences in chimpanzee and macaque genomes (panTro3 and rheMac2, respectively) corresponding to human precursors using BLAT (Kent 2002) and then aligned these by ClustalW2 (http://www.ebi.ac.uk/Tools/msa/clustalw2) to extract the orthologous mature sequences. The processed miRNA datasets are available as supplementary files on the Series record.