Computational protocol: Decreased Expression of Synapse-Related Genes and Loss of Synapses in Major Depressive Disorder

Similar protocols

Protocol publication

[…] An aliquot of the total RNA that was previously extracted from prefrontal cortex punches was used for secondary validation using real time PCR. Five hundred ng of total RNA was used for cDNA synthesis using oligo dT primers and SuperScript II reverse transcriptase (Invitrogen), and subsequently diluted with nuclease-free water to 10 ng ul−1 cDNA. Gene-specific high-melt temperature primers for genes of interest were designed using Primer 3 software (http://frodo.wi.mit.edu/cgi-bin/primer3/primer3_www.cgi; Whitehead Institute for Biomedical Research) and expressed sequence information obtained from GenBank (NCBI). PCR reactions were conducted on an ABI 7900 Sequence Detection System (Applied Biosystems) using a hotstart SYBR-green based method (Quantitect, QIAGEN) followed by melt curve analysis to verify specificity of product. The CT value (cycle number at threshold) was used for calculations of relative amount of mRNA molecules. The CT value of each target genes was normalized by subtraction of the CT value from multiple housekeeping genes. This value is the ΔCT. The difference in ΔCT between Control and MDD represents the ΔΔCT, and the relative quantitative change was showed as 2−ΔΔCT. All of the genes of interest were normalized to the housekeeping gene, cyclophilin. [...] The scoring matrix of TRANSFAC (http://www.biobase-international.com) was used for the binding motif search in promoter regions (1 kb of the 5’-flanking sequence and 200 bp on downstream from transcription start site) of our candidate genes. The over-represented TFBSs were determined in the set of promoters with the parameter of exact matches of core sequences (Core Score = 1) at 0.95 matrix similarity cut-off (Matrix Score ≥ 0.95) in the vertebrate matrix. […]

Pipeline specifications

Software tools Primer3, Biobase
Databases TRANSFAC
Applications Miscellaneous, qPCR
Organisms Rattus norvegicus, Homo sapiens