Testing for Allele-specific Expression from Human Brain Samples.
Testing for Allele-specific Expression from Human Brain Samples.
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DOI：
10.21769/bioprotoc.4832
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发表时间：
2023-10-05
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影响因子：
0.8
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全基因组关联研究鉴定出的许多单核苷酸多态性 (SNP) 通过等位基因特异性表达 (ASE) 作为表达数量性状位点 (eQTL) 对疾病风险产生影响。虽然存在用于探测正常个体组织中的 eQTL 的数据库，但人们可能希望确定这些数据库中未表征的特定组织或疾病状态中的 eQTL 或 ASE。在这里，我们提出了一个协议，用于评估已知全基因组关联研究（GWAS）帕金森病（PD）风险基因座的两个可能目标基因（GPNMB和KLHL7）的ASE，这些目标基因来自PD和神经正常个体的死后人脑组织。这是通过一系列 RNA 分离、cDNA 文库生成、使用可定制 cDNA 捕获探针富集感兴趣转录本、双端 RNA 测序和后续分析来完成的。相对于传统的基于批量 RNAseq 的方法，该方法提供了更高的灵敏度，并且提供了可扩展到其他基因、组织和疾病状态研究的蓝图。 主要特点 • 分析认知正常对照 (NC) 和帕金森病 (PD) 个体的脑裂解液中 GPNMB 等位基因特异性表达 (ASE)。 • 基于Mayba 等人的ASE 协议构建。 （2014）并将应用从细胞扩展到人体组织。 • 通过RNA CaptureSeq 富集所需转录本来提高灵敏度。 • 针对扣带回、尾状核和小脑的人脑裂解物进行了优化。
Many single nucleotide polymorphisms (SNPs) identified by genome-wide association studies exert their effects on disease risk as expression quantitative trait loci (eQTL) via allele-specific expression (ASE). While databases for probing eQTLs in tissues from normal individuals exist, one may wish to ascertain eQTLs or ASE in specific tissues or disease-states not characterized in these databases. Here, we present a protocol to assess ASE of two possible target genes (GPNMB and KLHL7) of a known genome-wide association study (GWAS) Parkinson’s disease (PD) risk locus in postmortem human brain tissue from PD and neurologically normal individuals. This was done using a sequence of RNA isolation, cDNA library generation, enrichment for transcripts of interest using customizable cDNA capture probes, paired-end RNA sequencing, and subsequent analysis. This method provides increased sensitivity relative to traditional bulk RNAseq-based and a blueprint that can be extended to the study of other genes, tissues, and disease states. Key features • Analysis of GPNMB allele-specific expression (ASE) in brain lysates from cognitively normal controls (NC) and Parkinson’s disease (PD) individuals. • Builds on the ASE protocol of Mayba et al. (2014) and extends application from cells to human tissue. • Increased sensitivity by enrichment for desired transcript via RNA CaptureSeq. • Optimized for human brain lysates from cingulate gyrus, caudate nucleus, and cerebellum.