Gene expression data from Guthrie blood-spot cards. Homo sapiens

Background: Standard Guthrie cards have been widely used to collect blood samples from neonates for newborn screening programs, and to a lesser extent, from normal controls and patients in research studies. Ease of blood collection (small quantity and less pain), transportation, and storage are the advantages of using these cards. It is believed that RNA obtained from these samples is of low quantity and degraded quality. However, we recently discovered that approximately 3,500 expressed genes can be detected from blood spot samples using in-house made, low resolution cDNA microarrays. Here, we established a new and improved methodology to acquire gene expression profiles from blood spot cards using commercially-available high resolution microarrays. We determined the optimal number of blood spot punches required for maximal RNA extraction, eliminated uses of trizol and chloroform for RNA extraction by using a modified protocol of the illustra Mini Spin Kit from GE-Whatm an, concentrated the quantity of RNA templates before amplification, improved amplification efficiency using the new Ribo-SPIA technology in WT-Ovation Pico System (WT-Pico) by NuGEN, before the samples were hybridized onto 4x44K whole human genome gene expression microarrays from Agilent. Nine dried blood spot samples were collected from a control population and stored at ~ -80 °C between 6 months to 2 years. High quality RNA was extracted from the buffy coat of the same individuals as a reference and processed using the standard Agilent microarray procedure. Commercially-available brain RNA was used as a positive control in both standard and new procedures for microarrays. Results: Three 3-mm punches produced the highest yield of total RNA using the non-trizol extraction method. Three to six nanogram per microliter of RNA can be concentrated and is sufficient to be amplified using the WT-Pico. Approximately 9,000 expressed genes can be detected after normalization and background correction of the microarray data. Conclusion: Genome-wide gene expression profile can be obtained from archived dried blood spot samples. Our new and improved methodology will add value to the perception of utilizing archival Guthrie cards eg. neonatal blood spot cards as unique biospecimens for molecular genomics and diagnostic studies of perinatal diseases such as pediatric cancers. Keywords: Gene Expression experiment Overall design: Archival guthrie blood-spot cards may contain valuable data for epidemiological or other studies. Showing microarray data from guthrie blood-spot cards

背景：标准格思里采血卡（Guthrie card）已广泛应用于新生儿筛查项目的新生儿血样采集；在科研研究中，其多用于采集正常对照及患者样本，应用相对较少。该类采血卡的优势在于采血便捷（采血量少、痛感低）、便于运输与储存。过往研究普遍认为，此类样本提取的核糖核酸（RNA）量少且降解严重。但本团队近期通过自制低分辨率互补脱氧核糖核酸（cDNA）微阵列，从血斑样本中检测到约3500个表达基因。
本研究建立了一套全新优化的方法，可通过商业化高分辨率微阵列从血斑采血卡中获取基因表达谱：本研究确定了实现最大RNA提取量的最佳血斑打孔数量；采用GE-Whatman公司的illustra Mini Spin Kit改良方案，无需使用TRIzol试剂与三氯甲烷（chloroform）完成RNA提取；在扩增前对RNA模板进行浓度富集；并借助NuGEN公司WT-Ovation Pico系统（WT-Pico）中的新型Ribo-SPIA技术提升扩增效率，随后将样本与安捷伦（Agilent）公司的4×44K全人类基因组基因表达微阵列进行杂交。
本研究从对照人群中收集了9份干血斑样本，于-80℃左右保存6个月至2年不等。从同一受试者的血沉棕黄层（buffy coat）中提取高质量RNA作为参照样本，并按照安捷伦标准微阵列流程进行处理。商业化售卖的脑组织RNA被用作两种微阵列实验流程（标准流程与新方法）的阳性对照。
结果：采用非TRIzol提取方法时，3枚3mm直径的血斑打孔样本的总RNA得率最高。可将RNA浓度富集至3~6 ng/μL，该浓度足以通过WT-Pico完成扩增。对微阵列数据进行归一化与背景校正后，可检测到约9000个表达基因。
结论：可从存档干血斑样本中获取全基因组范围的基因表达谱。本研究建立的新型优化方法，将推动存档格思里采血卡（例如新生儿血斑卡）作为独特生物样本，用于分子基因组学研究及围产期疾病（如儿童肿瘤）的诊断研究。
关键词：基因表达实验
整体设计：存档格思里血斑卡可为流行病学及其他研究提供宝贵数据。本数据集展示了格思里血斑卡的微阵列实验数据。