The effect of cell isolation methods on the human transcriptome profiling and microbial transcripts of peripheral blood.

The expression of human and microbial genes serves as biomarkers for disease and health. Blood RNA is an important biological resource for precision medicine and translational medicine. However, few studies have assessed the human transcriptome profiles and microbial communities composition and diversity of peripheral blood from different cell isolation methods, which could affect the reproducibility of researches. We collected peripheral blood from three healthy donors and processed it immediately. We used RNA sequencing to investigate the effect of three leukocyte isolation methods including buffy coat (BC) extraction, red blood cell (RBC) lysis and peripheral blood mononuclear cell (PBMC) isolation with the comparison with whole blood (WB), through analyzing the sensitivity of gene detection, the whole transcriptome profiling and microbial composition and diversity. Our data showed that BC extraction with high globin mRNA mapping rate had similar transcriptome profiles with WB, while RBC lysis and PBMC isolation depleted RBCs effectively. With the efficient depletion of RBC and distinct compositions of leukocyte subsets, RNA-seq of RBC lysis and PBMC isolation uniquely detected genes from specific cell types, like granulocytes and NK cells. In addition, we observed that the microbial composition and diversity were more affected by individuals than isolation methods. Our results showed that blood cell isolations could largely influence the sensitivity of detection of human genes and transcriptome profile.

Impact of storage conditions on peripheral leukocytes transcriptome.

Leukocytes reflect the physiological and pathological states of each individual, and transcriptomic data of leukocytes have been used to reflect health conditions. Since the overall impact of ex vivo conditions on the leukocyte transcriptome before RNA stabilization remains unclear, we evaluated the influence of temporary storage conditions on the leukocyte transcriptome through RNA sequencing. We collected peripheral blood with EDTA tubes, which were processed immediately or stored either at 4 °C or room temperature (RT, 18-22 °C) for 2 h, 6 h and 24 h. Total cellular RNA was extracted from 42 leukocyte samples after red blood cells lysis for subsequent RNA sequencing. We applied weighted gene co-expression network analysis to construct co-expression networks of mRNA and lncRNA among the samples, and then performed gene ontology (GO) term enrichment to explore possible biological processes affected by storage conditions. Storage conditions change the gene expression of peripheral leukocytes. Comparing with fresh leukocytes, storage for 24 h at 4 °C and RT affected 1515 (1.51%) and 10,823 (10.82%) genes, respectively. Pathway enrichment analysis identified nucleosome assembly enriched in up-regulated genes at both conditions. When blood was stored at RT for 24 h, genes involved in apoptotic signaling pathway, negative regulation of cell cycle and lymphocyte activation were upregulated, while the relative proportion of neutrophils was significantly decreased. Temporary storage conditions profoundly affect the gene expression profiles of leukocytes and might further change cell viability and state. Storage of blood samples at 4 °C within 6 h largely maintains their original transcriptome.

A Chinese patient with Rothmund-Thomson syndrome.

INTRODUCTION: Rothmund-Thomson syndrome (RTS) is a rare autosomal recessive disorder that has been reported in all ethnicities, with several identifiable pathogenic variants. There have been reported cases indicating that RTS may lead to low birth weight in fetuses, but specific data on the fetal period are lacking. Genetic testing for RTS II is currently carried out by identifying pathogenic variants in RECQL4. METHODS: In order to determine the cause, we performed whole-genome sequencing (WGS) analysis on the patient and his parents. Variants detected by WGS were confirmed by Sanger sequencing and examined in family members. RESULTS: After analyzing the WGS data, we found a heterozygous nonsense mutation c.2752G>T (p.Glu918Ter) and a novel frameshift insertion mutation c.1547dupC (p.Leu517AlafsTer23) of RECQL4, which is a known pathogenic/disease-causing variant of RTS. Further validation indicated these were compound heterozygous mutations from parents. CONCLUSION: Our study expands the mutational spectrum of the RECQL4 gene and enriches the phenotype spectrum of Chinese RTS patients. Our information can assist the patient's parents in making informed decisions regarding their future pregnancies. This case offers a new perspective for clinicians to consider whether to perform prenatal diagnosis.

Evaluating the Effects of Storage Conditions on Multiple Cell-Free RNAs in Plasma by High-Throughput Sequencing.

Background: Plasma cell-free RNAs (cfRNAs) can serve as noninvasive biomarkers for the diagnosis and monitoring of diseases. However, the delay in blood processing may lead to unreliable results. Therefore, an unbiased evaluation based on the whole transcriptome under different storage conditions is needed. Methods: Here, blood samples were collected in ethylenediaminetetraacetic acid tubes and processed immediately (0 hour), or stored at room temperature (RT) or 4°C for different time intervals (2, 6, and 24 hours) before plasma separation. High-throughput sequencing was applied to assess the effects of storage conditions on the transcript profiles and fragment characteristics of plasma cell-free mRNA, long noncoding RNA (lncRNA), and small RNAs. Results: More genes changed their expression levels with time when blood was stored at RT compared with those at 4°C. Cell-free mRNA and lncRNA were relatively stable in blood preserved at 4°C for 6 hours, while cell-free microRNA (miRNA) and piwi-interacting RNA (piRNA) remained stable at 4°C for 24 hours. After 24 hours, more contamination of the leukocyte-derived RNAs occurred at RT, possibly due to apoptosis. Meanwhile, significant changes were also observed regarding the characteristics of the RNA fragments, including fragment size, the proportion of intron, and the pyrimidine frequency of the fragmented 3' end. Fifteen tissue-enriched genes were detected in the plasma but not expressed in leukocytes. The expression level and fragment length of these genes gradually decreased during storage, suggesting the degradation of the cfRNA and the dilution of leukocyte-derived RNA with other tissue-derived cfRNA. Conclusions: Our results suggest that the contamination of leukocyte-derived RNA and the degradation of original cfRNA contribute to the changes in the cfRNA expression profiles and the fragment characteristics during short-term storage. The storage of blood at 4°C for 6 hours allows plasma cfRNA to remain relatively stable, which will be useful for further studies or clinical applications where adequate quantification or the fragment signature of cfRNA is required.

A Nested Case-Control Study of the Relationship between Salivary Inflammatory Mediators, Periodontal Parameters, and Preterm Birth in a Chinese Population.

Background: To explore whether salivary inflammatory mediators and periodontal indices at different gestational stages can be taken as indicators of preterm birth (PTB). Methods: This nested case-control study enrolled systemically healthy pregnant women at 9 to 36 weeks of gestation. Periodontal indices were measured at the enrollment date, and interleukin-1ß (IL-1ß), IL-6, tumor necrosis factor (TNF-α), prostaglandin E2 (PGE2), and 8-hydroxy-deoxyguanosine (8-OHdG) in the saliva were determined by enzyme-linked immunosorbent assay (ELISA). The birth outcome was recorded. Results: PTB occurred in 26 women. A total of 104 matched women with full-term birth (FTB) were used as controls. The PTB women enrolled at 24-28 gestational weeks displayed a significantly greater bleeding index (BI), probing pocket depth (PD), PD ≥ 4 mm sites (%), saliva-TNF-α, and saliva-PGE2 (P < 0.05). BI and PGE2 in the saliva were found to be positively associated with PTB (OR = 4.79, P = 0.048, 95%CI = 1.014 to 22.628; OR = 1.07, P = 0.04, 95%CI = 1.004 to 1.135, respectively). The areas under the receiver operating characteristic curve (ROC) of BI and saliva-PGE2 were 0.82 and 0.78, respectively, and that of the combined detection was 0.91, which was larger than either marker alone, although the differences were not significant (P > 0.05). Conclusions: The combination of BI and PGE2 in saliva at 24-28 gestational weeks could be a predictor of PTB in asymptomatic women. However, the results should be further explored with larger sample size.

Polyadenylation ligation-mediated sequencing (PALM-Seq) characterizes cell-free coding and non-coding RNAs in human biofluids.

BACKGROUND: Cell-free messenger RNA (cf-mRNA) and long non-coding RNA (cf-lncRNA) are becoming increasingly important in liquid biopsy by providing biomarkers for disease prediction, diagnosis and prognosis, but the simultaneous characterization of coding and non-coding RNAs in human biofluids remains challenging. METHODS: Here, we developed polyadenylation ligation-mediated sequencing (PALM-Seq), an RNA sequencing strategy employing treatment of RNA with T4 polynucleotide kinase to generate cell-free RNA (cfRNA) fragments with 5' phosphate and 3' hydroxyl and RNase H to deplete abundant RNAs, achieving simultaneous quantification and characterization of cfRNAs. RESULTS: Using PALM-Seq, we successfully identified well-known differentially abundant mRNA, lncRNA and microRNA in the blood plasma of pregnant women. We further characterized cfRNAs in blood plasma, saliva, urine, seminal plasma and amniotic fluid and found that the detected numbers of different RNA biotypes varied with body fluids. The profiles of cf-mRNA reflected the function of originated tissues, and immune cells significantly contributed RNA to blood plasma and saliva. Short fragments (<50 nt) of mRNA and lncRNA were major in biofluids, whereas seminal plasma and amniotic fluid tended to retain long RNA. Body fluids showed distinct preferences of pyrimidine at the 3' end and adenine at the 5' end of cf-mRNA and cf-lncRNA, which were correlated with the proportions of short fragments. CONCLUSION: Together, PALM-Seq enables a simultaneous characterization of cf-mRNA and cf-lncRNA, contributing to elucidating the biology and promoting the application of cfRNAs.

Association between periodontal indexes and biomarkers in gingival crevicular fluid and preterm birth in pregnancy: a nested case-control study.

OBJECTIVES: This study aimed to investigate the association between periodontal indexes and biomarkers in gingival crevicular fluid (GCF) and preterm birth (PTB) in pregnancy, as well as to assess the clinical value of these indexes as predictors of PTB. METHODS: A nested case-control study was conducted. A total of 300 systematically healthy pregnant women were selected within 36 weeks of gestation and grouped according to the enrolled weeks. Periodontal indexes, including probing depth (PD), bleeding index (BI), gingival index (GI), and five biomarkers in GCF, including interleukin (IL)-1ß, IL-6, tumor necrosis factor-α (TNF-α), prostaglandin E2 (PGE2), and 8-hydroxy-2-deoxyguanosine (8-OHdG) were measured at the enrolled date. The detailed birth outcome was recorded. RESULTS: Only women at 24-28 weeks of gestation per PTB case (four full-term births) were selected as controls subjects, PTB displayed significantly greater GI, BI, and 8-OHdG (P<0.05). Logistic regression analysis revealed that BI and 8-OHdG were the dependent risk factors of PTB (OR=5.90, P=0.034; OR=1.18, P=0.045, respectively). The areas under the receiver operating characteristic curve (ROC) of BI and 8-OHdG were 0.80 and 0.69, and that of the combined detection was 0.82, which was larger than the individual detection, although the differences were not significant (P>0.05). CONCLUSIONS: Increased BI and 8-OHdG at 24-28 weeks of gestation are risk factors for PTB. Their combined detection may have some value in the prediction of PTB, but further studies with a larger sample size are needed to explore it and thus provide experiment evidence for establishing an early warning system for PTB in pregnant women with periodontal disease.