Amplification and sequencing of 16S amplicons are widely used for profiling the structure of oral microbiota. However, it remains not clear whether and to what degree DNA extraction and targeted 16S rRNA hypervariable regions influence the analysis. Based on a mock community consisting of five oral bacterial species in equal abundance, we compared the 16S amplicon sequencing results on the Illumina MiSeq platform from six frequently employed DNA extraction procedures and three pairs of widely used 16S rRNA hypervariable primers targeting different 16S rRNA regions. Technical reproducibility of selected 16S regions was also assessed. DNA extraction method exerted considerable influence on the observed bacterial diversity while hypervariable regions had a relatively minor effect. Protocols with beads added to the enzyme-mediated DNA extraction reaction produced more accurate bacterial community structure than those without either beads or enzymes. Hypervariable regions targeting V3-V4 and V4-V5 seemed to produce more reproducible results than V1-V3. Neither sequencing batch nor change of operator affected the reproducibility of bacterial diversity profiles. Therefore, DNA extraction strategy and 16S rDNA hypervariable regions both influenced the results of oral microbiota biodiversity profiling, thus should be carefully considered in study design and data interpretation.

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Impact of DNA extraction method and targeted 16S-rRNA hypervariable region on oral microbiota profiling

Measuring coverage and accuracy of whole-exome sequencing in clinical context.

The present study aimed to construct a rat model of rheumatoid arthritis (RA) to evaluate changes in pathology, the expression of inflammatory factors and regulation of signaling pathways. The protective effect of tetrandrine (Tet) on tissue lesions induced by RA was also investigated. A total of 60 Wistar rats (100-200 g) were randomly divided into six groups (n=10 per group), namely a blank (NC) group, model group, methotrexate (MTX) group (3 mg/kg body weight), high-dose Tet group (31.25 mg/kg body weight), medium-dose Tet group (18.75 mg/kg body weight) and low-dose Tet group (6.25 mg/kg body weight). A rat model of RA was induced via injection of 0.1 ml complete Freund's adjuvant into the right rear toe. Toe swelling rate, arthritis index and immune organ index were calculated. In addition, cyclooxygenase (COX)-2 expression at the mRNA and protein level in the peripheral blood mononuclear cells (PBMCs) of rats were determined by reverse transcription-quantitative polymerase chain reaction and western blot analysis, respectively. Serum concentrations of inflammatory factors were measured using enzyme-linked immunosorbent assays. It was observed that treatment with Tet alleviated the severity of rear toe swelling associated with RA in rats. Furthermore, Tet exerted anti-inflammatory and immunosuppressive effects in the rat model of RA. Tet also reduced the expression of COX-2 in PBMCs and lowered the concentrations of inflammatory factors in the serum of RA rats. The present data indicate that Tet may exert pharmacological effects in the treatment of RA. The mechanism of action of Tet may be associated with the regulation of inflammatory factors and the inhibition of immune organs.

/pdf/tetrandrine-alleviates-symptoms-of-rheumatoid-arthritis-in-14l7e9am79.pdf

Tetrandrine alleviates symptoms of rheumatoid arthritis in rats by regulating the expression of cyclooxygenase‑2 and inflammatory factors

Conventional DNA sample preparation methods involve tedious sample handling steps that require numerous inhibitors of the polymerase chain reaction (PCR) and instrumentation to implement. These disadvantages limit the applicability of conventional cell lysis and DNA extraction methods in high-throughput applications, particularly in forensics and clinical laboratories. To overcome these drawbacks, a series of nine hydrophobic magnetic ionic liquids (MILs) previously shown to preconcentrate DNA were explored as cell lysis reagents. The MILs were found to lyse white blood cells from whole blood, 2-fold diluted blood, and dry blood samples while simultaneously extracting human genomic DNA. The identity of metal ion incorporated within the MIL appears to cause hemolysis while the cationic component further reduces the cell’s integrity. Over 500 pg of human genomic DNA was isolated from 50 μL of whole blood using the trioctylbenzylammonium tris(hexafluoroacetylaceto)nickelate(II) ([N8,8,8,Bz+][Ni(hfacac)3−]) MIL, and 800 pg DNA was isolated from a dry blood samples using the trihexyl(tetradecyl)phosphonium tris(phenyltrifluoroacetylaceto)nickelate(II) ([P6,6,6,14+][Ni(Phfacac)3−]) MIL following a 1-min vortex step. A rapid, one-step cell lysis and DNA extraction from blood is ideal for settings that seek high-throughput analysis while minimizing the potential for contamination. Graphical abstract

Simultaneous cell lysis and DNA extraction from whole blood using magnetic ionic liquids.

Chronic obstructive pulmonary disease (COPD) is a chronic lung inflammatory disease which has a close relationship with aging. Genome-wide analysis reveals that DNA methylation markers vary obviously with age. DNA methylation variations in peripheral blood have the potential to be biomarkers for COPD. However, the specific DNA methylation of aging-related genes in the peripheral blood of COPD patients remains largely unknown. Firstly, 9 aging-related differentially expressed genes (DEGs) in COPD patients were screened out from the 25 aging-related genes profile through a comprehensive screening strategy. Secondly, qPCR and multiple targeted bisulfite enrichment sequencing (MethTarget) were used to detect the mRNA level and DNA methylation level of the 9 differentially expressed genes in the peripheral blood of 60 control subjects and 45 COPD patients. The candidate functional CpG sites were selected on the basis of the regulation ability of the target gene expression. Thirdly, the correlation was evaluated between the DNA methylation level of the key CpG sites and the clinical parameters of COPD patients, including forced expiratory volume in one second (FEV1), forced expiratory volume in one second as percentage of predicted volume (FEV1%), forced expiratory volume/ forced vital capacity (FEV/FVC), modified British medical research council (mMRC) score, acute exacerbation frequency and the situation of frequent of acute aggravation (CAT) score. Lastly, differentially methylated CpG sites unrelated to smoking were also determined in COPD patients. Of the 9 differentially expressed aging-related genes, the mRNA expression of 8 genes were detected to be significantly down-regulated in COPD group, compared with control group. Meanwhile, the methylated level of all aging-related genes was changed in COPD group containing 219 COPD-related CpG sites in total. Notably, 27 CpG sites of FOXO3 gene showed a lower False Discovery Rate (FDR) and higher methylation difference values. Also, some variable DNA methylation is associated with the severity of COPD.
 Additionally, of the 219 COPD-related CpG sites, 147 CpG sites were not related to smoking. These results identified that the mRNA expression and DNA methylation level of aging-related genes were changed in male COPD patients, which provides a molecular link between aging and COPD. The identified CpG markers are associated with the severity of COPD and provide new insights into the prediction and identification of COPD.

/pdf/variable-dna-methylation-of-aging-related-genes-is-489p31qsqw.pdf

Variable DNA methylation of aging-related genes is associated with male COPD

The extraction of genomic DNA is the crucial first step in large-scale epidemiological studies. Though there are many popular DNA isolation methods from human whole blood, only a few reports have compared their efficiencies using both end-point and real-time PCR assays. Genomic DNA was extracted from coronary artery disease patients using solution-based conventional protocols such as the phenol-chloroform/proteinase-K method and a non-phenolic non-enzymatic Rapid-Method, which were evaluated and compared vis-a-vis a commercially available silica column-based Blood DNA isolation kit. The appropriate method for efficiently extracting relatively pure DNA was assessed based on the total DNA yield, concentration, purity ratios (A260/A280 and A260/A230), spectral profile and agarose gel electrophoresis analysis. The quality of the isolated DNA was further analysed for PCR inhibition using a murine specific ATP1A3 qPCR assay and mtDNA/Y-chromosome ratio determination assay. The suitability of the extracted DNA for downstream applications such as end-point SNP genotyping, was tested using PCR-RFLP analysis of the AGTR1-1166A>C variant, a mirSNP having pharmacogenetic relevance in cardiovascular diseases. Compared to the traditional phenol-chloroform/proteinase-K method, our results indicated the Rapid-Method to be a more suitable protocol for genomic DNA extraction from human whole blood in terms of DNA quantity, quality, safety, processing time and cost. The Rapid-Method, which is based on a simple salting-out procedure, is not only safe and cost-effective, but also has the added advantage of being scaled up to process variable sample volumes, thus enabling it to be applied in large-scale epidemiological studies.

Irin Kurikesu

Papers

Evaluating genomic DNA extraction methods from human whole blood using endpoint and real-time PCR assays.