Optimized protocol for the extraction of RNA and DNA from frozen whole blood sample stored in a single EDTA tube

Cryopreservation of whole blood is useful for DNA collection, and clinical and basic research. Blood samples in ethylenediaminetetraacetic acid disodium salt (EDTA) tubes stored at − 80 °C are suitable for DNA extraction, but not for high-quality RNA extraction. Herein, a new methodology for high-quality RNA extraction from human blood samples is described. Quickly thawing frozen whole blood on aluminum blocks at room temperature could minimize RNA degradation, and improve RNA yield and quality compared with thawing the samples in a 37 °C water bath. Furthermore, the use of the NucleoSpin RNA kit increased RNA yield by fivefold compared with the PAXgene Blood RNA Kit. Thawing blood samples on aluminum blocks significantly increased the DNA yield by ~ 20% compared with thawing in a 37 °C water bath or on ice. Moreover, by thawing on aluminum blocks and using the NucleoSpin RNA and QIAamp DNA Blood kits, the extraction of RNA and DNA of sufficient quality and quantity was achieved from frozen EDTA whole blood samples that were stored for up to 8.5 years. Thus, extracting RNA from frozen whole blood in EDTA tubes after long-term storage is feasible. These findings may help advance gene expression analysis, as well as biomarker research for various diseases

Interferon signaling and hypercytokinemia-related gene expression in the blood of antidepressant non-responders

Only 50% of patients with depression respond to the first antidepressant drug administered. Thus, biomarkers for prediction of antidepressant responses are needed, as predicting which patients will not respond to antidepressants can optimize selection of alternative therapies. We aimed to identify biomarkers that could predict antidepressant responsiveness using a novel data-driven approach based on statistical pattern recognition. We retrospectively divided patients with major depressive disorder into antidepressant responder and non-responder groups. Comprehensive gene expression analysis was performed using peripheral blood without narrowing the genes. We designed a classifier according to our own discrete Bayes decision rule that can handle categorical data. Nineteen genes showed differential expression in the antidepressant non-responder group (n = 15) compared to the antidepressant responder group (n = 15). In the training sample of 30 individuals, eight candidate genes had significantly altered expression according to quantitative real-time polymerase chain reaction. The expression of these genes was examined in an independent test sample of antidepressant responders (n = 22) and non-responders (n = 12). Using the discrete Bayes classifier with the HERC5, IFI6, and IFI44 genes identified in the training set yielded 85% discrimination accuracy for antidepressant responsiveness in the 34 test samples. Pathway analysis of the RNA sequencing data for antidepressant responsiveness identified that hypercytokinemia- and interferon-related genes were increased in non-responders. Disease and biofunction analysis identified changes in genes related to inflammatory and infectious diseases, including coronavirus disease. These results strongly suggest an association between antidepressant responsiveness and inflammation, which may be useful for future treatment strategies for depression

Novel endogenous retrovirus-derived transcript expressed in the bovine placenta is regulated by WNT signaling

Endogenous retroviruses (ERVs) are involved in placentation; perhaps, the most well-known ERVs are the syncytins, actively transcribed env genes involved in cell–cell fusion and possible morphological variations. However, ERVs other than syncytins that play an important role in placental development have not been well characterized. To identify ERV genes expressed during the onset of placentation in the bovine species, we characterized the expression profiles of bovine conceptus transcripts during the peri-attachment period using RNA-seq analysis, and confirming some candidates through real-time PCR. Using in silico and PCR analyses, we identified a novel ERV proviral sequence derived from a gag region, designated bovine endogenous retroviruses (BERV)-K3, containing Gag_p10 and Gag_p24, zinc finger domain. Initial expression of this ERV in bovine conceptuses was on day 20 (day 0 = day of estrus), soon after conceptus attachment to the endometrial epithelium, and its high placental expression was maintained up to the middle of pregnancy. The BERV-K3 transcript was also found in the uterine luminal and glandular epithelia, liver, kidney, intestine, and skin. BERV-K3 is located on chromosome 7 and integrated within LOC100848658, from which noncoding RNA could be transcribed. Furthermore, the expression of endogenous BERV-K3 in bovine trophoblast cell lines was induced by a WNT agonist, a signaling system common to genes expressed in placentas. These data support the argument that during the evolutionary process, mammals incorporated not only similar ERV sequences, but also ERVs unique to individual species. BERV-K3 is in the latter case, likely providing functions unique to ruminant gestation

Gene expression of CCL8 and CXCL10 in peripheral blood leukocytes during early pregnancy in cows

Abstract Background The aim of the present study was to evaluate CCL8 and CXCL10 expression and its regulatory mechanism in peripheral blood leukocytes (PBLs) at the time of maternal recognition in cows. Blood samples were collected on 14, 15, 16, 17 and 18 d after artificial insemination (AI). Based on the day of return of estrus, cows were divided into three groups, pregnant (n = 5), early embryonic mortality (EEM; n = 5) and late embryonic mortality (LEM; n = 5). The gene expression levels in PBLs were assessed with quantitative real-time reverse transcription PCR. Results The expression of CCL8 and CXCL10 mRNA in PBLs gradually increased from 14 to 18 d of pregnant cows and significant differences were observed on 18 d (P < 0.05), whereas no significant changes were observed both in EEM and LEM cows. Interferon-stimulated protein 15 kDa (ISG15), myxovirus-resistance gene (MX) 1 and MX2 mRNA expression in PBLs increased from 14 to 18 d which was significant on 18 d of pregnant cows as well as in LEM cows (P < 0.05), but no changes were observed in EEM cows. To determine whether the expression of CCL8 and CXCL10 in PBLs was regulated by pregnancy-related substances or not, expression level was assessed after exposure to interferon-τ (IFNT) and CCL16. Monocytes, granulocytes and lymphocytes were obtained using density-gradient centrifugation and flow cytometry. The addition of IFNT (100 ng/mL) and CCL16 (100 ng/mL) to cultured PBLs increased the expression of CCL8 and CXCL10 mRNA (P < 0.05). The expression of ISG15, MX1 and MX2 mRNA in PBLs was also stimulated by IFNT and CCL16 (P < 0.05). Conclusions The expression of CCL8 and CXCL10 genes increased in PBLs during early pregnancy. Since IFNT stimulated CCL8 and CXCL10 expression in cultured PBLs, the increase of CCL8 and CXCL10 might be pregnancy-dependent events. The expression of both CCL8 and CXCL10 in PBLs was stimulated by CCL16 as well as IFNT, suggesting a chemokine interaction that at least includes CCL8, CXCL10 and CCL16, and may play a role in regulating maternal recognition in cows