DNA and RNA samples from blood are the common examination target for non-invasive physical tests and/or biomedical studies. Since high-quality DNA and RNA samples guarantee the correctness of these tests and/or studies, we investigated the effects of storage temperature and storage duration of whole blood on DNA and RNA qualities. Subjects were enrolled to donate blood samples which were stored for different durations and at different temperatures, followed by the examinations on RNA quality, qPCR, DNA quality and DNA methylation. For RNA, we observed obvious quality decline with storage duration longer than 24 hours. Storage at low temperature does not keep RNA samples from degradation. And, storing whole blood samples in freezer dramatically damage RNA. For DNA, quality decline was not observed even with storage duration for 15 days. However, DNA methylation significantly altered with storage duration longer than three days. Storage duration within 24 hours is critical for collecting high-quality RNA samples for next-generation sequencing (NGS) assays (RIN≧8). If microarray assays are expected (RIN≧7), storage duration within 32 hours is acceptable. Although DNA is resistant within 15 days when kept in whole blood, DNA quantity dramatically decreases owing to WBC lysis. In addition, duration for more than three days significantly alter DNA methylation status, globally and locally. Our result provides a reference for dealing with blood samples.

This study investigated whether common comorbidities or biochemical factors, such as allergic disease, anemia, inflammation, and neurotransmitters, are singly or additively associated with an increased risk of attention deficit–hyperactivity disorder (ADHD). We recruited 216 children diagnosed with ADHD and 216 age-, sex-, height-, weight-, and class-matched controls from 31 elementary schools in Taipei, Taiwan. The International Study of Asthma and Allergies in Childhood questionnaire was used to measure allergic symptoms. Fasting venous blood was collected and analyzed for complete blood count, white blood cell differential count, immunoglobulin (Ig) E level, and serotonin (5-HT) level. The results showed that symptoms of both rhinitis (OR = 2.08, 95% CI = 1.42–3.05) and eczema (OR = 1.72, 95% CI = 1.02–2.88) were significantly associated with increased risk of ADHD. Children with ADHD showed considerably lower levels of hemoglobin (p = 0.001) and 5-HT (p < 0.001) and higher IgE level (p < 0.001) and eosinophil count (p = 0.001) than did control children. ADHD risk increased with the number of aforementioned biochemical risk factors present (one factor: OR = 1.87, 95% CI = 0.87–4.18; two factors: OR = 2.90, 95% CI = 1.29–6.48; three factors: OR = 4.47, 95% CI = 1.97–10.13; four factors: OR = 6.53, 95% CI = 2.43–17.57). Findings suggest that either ADHD’s etiology is multidimensional or the aforementioned conditions have shared etiology with ADHD.

Attention-deficit/hyperactivity disorder (ADHD) is a common childhood mental disorder with undetermined pathophysiological mechanisms. The gut microbiota and immunological dysfunction may influence brain functions and social behaviours. In the current study, we aimed to explore the correlation of gut microbiome imbalance and inflammation in the pathophysiology of ADHD. Forty-one children with ADHD and thirty-nine healthy-control (HC) individuals were recruited. Faecal samples from all participants were collected and submitted for 16 S rRNA V3–V4 amplicon microbiome sequencing analysis. The plasma levels of 10 cytokines, including TNF-α, IL-6, IL-1β, IL-2, IL-10, IL-13, IL-17A, IFN-α2, IFN-γ, and MCP-1, were determined using a custom-made sandwich enzyme-linked immunosorbent assay (ELISA) developed by Luminex Flowmetrix. There was no significant difference between the ADHD and HC groups in species diversity in the faeces, as determined with α-diversity and β-diversity analysis. In the ADHD group, three differentially abundant taxonomic clades at the genus level were observed, namely Agathobacter, Anaerostipes, and Lachnospiraceae. Top differentially abundant bacteria and representative biological pathways were identified in children with ADHD using linear discriminant analysis (LDA) effect size (LEfSe), and the phylogenetic investigation of communities by reconstruction of unobserved states (PICRUSt) analysis, respectively. The plasma levels of TNF-α were significantly lower in children with ADHD than in HCs. Within the ADHD group, the levels of TNF-α were negatively correlated with ADHD symptoms and diversity of the gut microbiome. Our study provides new insights into the association between gut microbiome dysbiosis and immune dysregulation, which may contribute to the pathophysiology of ADHD.