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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.

A form of systemic vasculitis, Kawasaki disease (KD) occurs most frequently in children under the age of five years old. Previous studies have found that Prostaglandin E2 (PGE2) correlates with KD, although the related mechanisms are still unknown. CD40L may also be a marker of vasculitis in KD, so this study focuses on PGE2 and CD40L expression in KD.This study consisted of a total of 144 KD patients, whose intravenous immunoglobulin (IVIG)/coronary arterial lesion (CAL) formation resistance was evaluated. PGE2 levels were evaluated in vitro to study the effect of CD40L on CD4+ T lymphocytes.PGE2 levels significantly increased after IVIG treatment (p

Kawasaki disease (KD) or Kawasaki syndrome is known as a vasculitis of small to medium-sized vessels, and coronary arteries are predominantly involved in childhood. Generally, 20-25% of untreated with IVIG and 3-5% of treated KD patients have been developed coronary artery lesions (CALs), such as dilatation and aneurysm. Understanding how coronary artery aneurysms (CAAs) are established and maintained in KD patients is therefore of great importance. Upon our previous genotyping data of 157 valid KD subjects, a genome-wide association study (GWAS) has been conducted among 11 (7%) CAA-developed KD patients to reveal five significant genetic variants passed pre-defined thresholds and resulted in two novel susceptibility protein-coding genes, which are NEBL (rs16921209 (P = 7.44 × 10(-9); OR = 32.22) and rs7922552 (P = 8.43 × 10(-9); OR = 32.0)) and TUBA3C (rs17076896 (P = 8.04 × 10(-9); OR = 21.03)). Their known functions have been reported to associate with cardiac muscle and tubulin, respectively. As a result, this might imply their putative roles of establishing CAAs during KD progression. Additionally, various model analyses have been utilized to determine dominant and recessive inheritance patterns of identified susceptibility mutations. Finally, all susceptibility genes hit by significant genetic variants were further investigated and the top three representative gene-ontology (GO) clusters were regulation of cell projection organization, neuron recognition, and peptidyl-threonine phosphorylation. Our results help to depict the potential routes of the pathogenesis of CAAs in KD patients and will facilitate researchers to improve the diagnosis and prognosis of KD in personalized medicine.

Kawasaki disease (KD) is a systemic vasculitis primarily affecting children < 5 years old. Genes significantly associated with KD mostly involve cardiovascular, immune, and inflammatory responses. Recent studies have observed stronger associations for KD risk with multiple genes compared to individual genes. Therefore, we investigated whether gene combinations influenced KD susceptibility or coronary artery lesion (CAL) formation. We examined 384 single-nucleotide polymorphisms (SNPs) for 159 immune-related candidate genes in DNA samples from KD patients with CAL (n = 73), KD patients without CAL (n = 153), and cohort controls (n = 575). Individual SNPs were first assessed by univariate analysis (UVA) and multivariate analysis (MVA). We used multifactor dimensionality reduction (MDR) to examine individual SNPs in one-, two-, and three-locus best fit models. UVA identified 53 individual SNPs that were significantly associated with KD risk or CAL formation (p < 0.10), while 35 individual SNPs were significantly associated using MVA (p ≤ 0.05). Significant associations in MDR analysis were only observed for the two-locus models after permutation testing (p ≤ 0.05). In logistic regression, combined possession of PDE2A (rs341058) and CYFIP2 (rs767007) significantly increased KD susceptibility (OR = 3.54; p = 4.14 x 10(-7)), while combinations of LOC100133214 (rs2517892) and IL2RA (rs3118470) significantly increased the risk of CAL in KD patients (OR = 5.35; p = 7.46 x 10(-5)). Our results suggest varying gene-gene associations respectively predispose individuals to KD risk or its complications of CAL.

MicroRNAs (miRNAs) play crucial roles in various physiological processes through post-transcriptional regulation of gene expressions and are involved in development, metabolism, and many other important molecular mechanisms and cellular processes. The Bombyx mori genome sequence provides opportunities for a thorough survey for miRNAs as well as comparative analyses with other sequenced insect species.We identified 114 non-redundant conserved miRNAs and 148 novel putative miRNAs from the B. mori genome with an elaborate computational protocol. We also sequenced 6,720 clones from 14 developmental stage-specific small RNA libraries in which we identified 35 unique miRNAs containing 21 conserved miRNAs (including 17 predicted miRNAs) and 14 novel miRNAs (including 11 predicted novel miRNAs). Among the 114 conserved miRNAs, we found six pairs of clusters evolutionarily conserved cross insect lineages. Our observations on length heterogeneity at 5' and/or 3' ends of nine miRNAs between cloned and predicted sequences, and three mature forms deriving from the same arm of putative pre-miRNAs suggest a mechanism by which miRNAs gain new functions. Analyzing development-related miRNAs expression at 14 developmental stages based on clone-sampling and stem-loop RT PCR, we discovered an unusual abundance of 33 sequences representing 12 different miRNAs and sharply fluctuated expression of miRNAs at larva-molting stage. The potential functions of several stage-biased miRNAs were also analyzed in combination with predicted target genes and silkworm's phenotypic traits; our results indicated that miRNAs may play key regulatory roles in specific developmental stages in the silkworm, such as ecdysis.Taking a combined approach, we identified 118 conserved miRNAs and 151 novel miRNA candidates from the B. mori genome sequence. Our expression analyses by sampling miRNAs and real-time PCR over multiple developmental stages allowed us to pinpoint molting stages as hotspots of miRNA expression both in sorts and quantities. Based on the analysis of target genes, we hypothesized that miRNAs regulate development through a particular emphasis on complex stages rather than general regulatory mechanisms.