High-throughput Sequencing Was Used To Measure The MRNA Imbalance Of Candidate Genes In The Human Brain Cortex

[Objective] Develop a high-throughput assay for measuring allelic expression imbal-ance (AEI) of mRNAs in human brain.[Methods]1) Compile a list of genes related to the major neuropsychiatric disorders:i) schizophrenia/autism, ii) Alzhheimer’s dis-ease and iii) heroin addiction with mRNA s that contain one or more single nucleotide polymorphisms (SNPs) with high heterozygosity in the Han Chinese population.2) Extract genomic DNA (gDNA) and total RNA from human prefrontal cortex (Brodmann area46) and reverse-transcribe the mRNA into cDNA.3) PCR amplify fragments of gDNA and cDNA that harbor high-heterozygosisty SNPs for XX candi-date genes and, ligate to brain sample-specific index-adapters.4) Pool PCR products and sequence on Solexa Sequencing Platform.5) Sort DNA sequences according to:i) brain sample, ii) gene and iii) allele.6) For each gene in each brain sample:count the number of sequences obtained for each allele and calculate the ratio of these numbers.[Results] We tested this method using a set73candidate genes, and obtained excel-lents results from71. AEI measurements of showed higher reproducibility and accu-racy compared to a previously developed low-throughput method.[Conclusions] This method is a ideal way to measure allelic expression at medium-to-high throughput.