Breast milk, as the optimal food for infants and young children, contains all the components necessary for proper growth and development. It is a rich source of both essential nutrients and biologically active factors, making breast milk a unique food with scientifically proven health-promoting properties. Among the entire range of biologically active factors, breast milk microorganisms and prebiotic factors, in the form of breast milk oligosaccharides, occupy an important place. The aim of our research was to determine the occurrence of bacteria with probiotic potential, belonging to the Lactobacillaceae family, in the environment of breast milk and breast milk oligosaccharides. The study included 63 human milk samples from breastfeeding women at various stages of lactation. Microorganism identification based on culture tests and MALDI TOF/MS, macronutrient analysis using the MIRIS human milk analyser, as well as analysis of human milk oligosaccharides using ultra-high-performance liquid chromatography coupled with high-resolution mass spectrometry were performed. The results have shown that breast milk from different breastfeeding women is characterized by great diversity in terms of the presence of Lacto-bacillaceae bacteria in its microbiological composition. These bacteria were present in 22.2 % of the tested breast milk samples. Analysis of the human milk oligosaccharide profile revealed a slightly higher content of prebiotic factors in breast milk samples containing Lactobacillaceae, including 2'-fucosyllactose, oligosaccharide occurring in the highest amount in breast milk.
Although next-generation RNA sequencing (RNA-seq) is increasingly incorporated into germline cancer predisposition testing, its diagnostic utility is often limited by low expression of many clinically relevant genes. To improve RNA yield and transcript representation for targeted RNA-seq, we optimized a simple protocol based on short-term lymphocyte culture prepared directly from whole blood collected in Li-heparin tubes. We systematically evaluated biological reproducibility and pre-analytical sample handling variability and demonstrated that whole blood can be stored at 4 °C for up to 5 days prior to lymphocyte cultivation without compromising RNA quality/gene expression. Gene expression was comparable for RNA isolated from K2EDTA and Tempus tubes, whereas short-term lymphocyte culture resulted in a substantial increase in expression of clinically important genes including BRCA1, BRCA2, RAD51C, RAD51D, PALB2, CHEK2, and multiple Fanconi anaemia genes otherwise low expressed in whole blood. Cultivation for 3-5 days did not significantly affect lymphocyte gene expression, providing flexibility for routine dia-gnostics. The protocol also enables inhibition of nonsense-mediated decay to facilitate analysis of variants causing premature termination. As a proof of principle, we characterized the splicing impact of FANCA c.2602-3C>G variant (located in intron 27) using cultured lymphocytes from its carrier. The variant causes deletion of six nucleotides in the mature transcript (ΔE28p(-6)/r.2602_2607del), resulting in an in-frame deletion (p. Gln869_Phe870del). This spliceogenic effect was reliably detectable only in cultured lymphocytes preferentially expressing the full-length FANCA transcript. Overall, short-term lymphocyte culture re-presents a simple and flexible RNA source that enhances variant interpretation in clinical RNA-seq analyses.
Lung adenocarcinoma is a prevalent cancer worldwide. MicroRNAs (miRNAs) are key regulators in various cancers, with miR-200a-5p emerging as a potential candidate due to its possible role in lung adenocarcinoma. However, its exact mechanisms of action remain unclear. We performed transcriptome sequencing of 32 clinical pairs to identify differentially expressed miRNAs. miR-200a-5p expression was validated in tissues and cell lines (H1975, A549) using bioinformatics and RT-qPCR. Biological effects were assessed via CCK8, EdU, Transwell, scratch assays, flow cytometry, and Western blotting (phosphorylated transducer and activator of transcription 3, fibronectin, matrix metallopeptidase 9, proliferating cell nuclear antigen, and Wnt pathway-related proteins). A protein-protein interaction network predicted downstream targets, focusing on the Wnt pathway. In vivo experiments, immunohistochemistry, and Western blotting evaluated tumour growth and Ki-67/Wnt protein expression. We found that miR-200a-5p was significantly up-regulated in lung adenocarcinoma tissues and cells, correlating with poor prognosis. Up-regulation enhanced proliferation, migration and invasion while inhibiting apoptosis in vitro. Down-regulation produced opposite effects. In vivo, miR-200a-5p promoted tumour growth via Wnt pathway activation. We conclude that miR-200a-5p acts as an oncogenic factor in lung adenocarcinoma by facilitating progression through the Wnt pathway. These findings suggest its potential as a therapeutic target and prognostic biomarker.
FMS-like tyrosine kinase 3 (FLT-3) mutations represent one of the most common genetic anomalies in acute myeloid leukaemia (AML), particularly in adults. The two most common types of mutations, internal tandem duplications (ITD) and tyrosine kinase domain (TKD) point mutations, facilitate uncontrolled cellular proliferation and unfavourable patient outcomes. These mutations are linked with a high relapse rate and shorter overall survival, highlighting the need for targeted therapies to be used. Recent advances in the discovery of new agents enabled incorporation of FLT-3 inhibitors into the frontline treatment regimen. First-generation inhibitors, such as midostaurin, provided the foundation for targeted therapy, while recently developed agents such as gilteritinib and quizartinib have shown more selectivity and demonstrated superior clinical efficiency and improved tolerability. This review discusses the significance of FLT-3 mutations, the evolution of targeted therapies, current treatment guidelines, and ongoing challenges such as resistance and high relapse rates. We also discuss the emerging combinations of therapies and novel agents currently in clinical trials that aim to overcome resistance and improve long-term outcomes for patients with FLT-3-mutated AML.
Nigella sativa black cumin oil (BCO) exhibits well-documented anti-inflammatory and antioxidant properties; however, the impact of extraction-related compositional variation on its cellular and molecular effects in stem cell systems remains insufficiently characterized. This study investigated the effects of cold-pressed (CP) and supercritical CO2 (ScCO2)-extracted BCO on the viability, inflammatory response and angiogenic potential of adipose-derived mesenchymal stem cells (ASCs). The oil composition and antioxidant activity were assessed using GC/MS and DPPH assays, respectively. ASCs were treated with different volumes of BCO, and cell viability was evaluated at 24, 48 and 72 h using the MTT assay. Anti-inflammatory effects were evaluated by real-time PCR analysis of IL6 and IL10 mRNA expression, while angiogenesis-related molecular responses were evaluated based on VEGF mRNA expression. Both CP and ScCO2-extracted BCO significantly increased ASC viability in a volume-dependent manner, with the highest viability consistently observed at 100 µl (P < 0.05). Treatment with 50 µl and 100 µl of either oil significantly reduced IL6 expression and concomitantly increased IL10 expression at 24 and 48 h (P < 0.05). VEGF expression was also significantly up-regulated at these time points, with ScCO2-extracted BCO inducing a more pronounced and sustained angiogenic response (P < 0.05). These findings indicate that BCO enhances ASC via-bility and molecular responses in a volume-dependent manner, while differences associated with extraction-related compositional variation may modulate inflammation- and angiogenesis-related molecular signalling in ASCs.
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The accession number in Fig. 1 was not presented correctly. The original article was published in Folia Biologica (Praha) Volume 71, No. 3 (2025), 123-139. https://doi.org/10.14712/fb2025071030123.
The present study was undertaken to provide complementary information on the cell cycle of leukaemic lymphoblasts and myeloblasts based on the computer-assisted maximal nuclear diameter measurements. The measurements were carried out in "one-size cell layer" loci of bone marrow smears of patients suffering from B-cell acute lymphoblastic leukaemia (B-ALL), acute myeloblastic leukaemia with minimal differentiation (M0 AML), acute myeloblastic leukaemia without maturation (M1 AML), acute myeloblastic leukaemia with maturation (M2 AML) and chronic myeloid leukaemia (CML). The maximal nuclear diameter was also measured in peripheral blood mature lymphocytes of B-cell chronic lymphocytic leukaemia (CLL), which are known to be in the G0/G1 phase of the cell cycle. In contrast, the maximal nuclear diameter in lymphoblasts of patients with B-ALL was larger and reflected the S and G2 cell cycle phase. The largest incidence of myeloblasts with the smallest maximal nuclear diameter was in M0 AML. The smaller incidence of such cells was also noted in patients with M1 AML. Myeloblasts with larger nuclear bodies were present in M2 AML and CML. Thus, post-mitotic myeloblasts in G0 and G1 phase were characteristic of M0 and to a smaller extent of M1 AML. Myeloblasts with larger maximal nuclear diameter reflecting the S and G2 phase were dominant in M2 AML and CML. In summary, the nuclear size heterogeneity of leukaemic lymphoblasts and myeloblasts in bone marrow smears depends on various phases of the cell cycle classified according to the maximal nuclear diameter.
Medulloblastoma (MB) in children is associated with distinct molecular subgroups, reflecting substantial biological heterogeneity. The presence of isocitrate dehydrogenase 1 (IDH1) and IDH2 mutations in paediatric MB has been rarely reported and not routinely investigated. Our study included 23 samples from paediatric patients diagnosed with MB. Hotspot alterations at codons IDH1 R132 and IDH2 R172 were examined using Sanger sequencing following polymerase chain reaction (PCR). The mean age of the patients was 10 years (SD: 4.25), comprising 17 males and 6 females. All cases exhibited classical histological features of MB. β-Catenin expression was observed in four cases (17.4 %), while 19 cases (82.6 %) showed no expression. No statistically significant differences in progression-free survival (PFS) were found between MBs with positive or negative β-catenin expression (P = 0.6). Radiotherapy alone was administered to four patients (17.4 %), while 19 patients (82.6 %) received combined radiotherapy and chemotherapy. The median PFS was 383 days (1 year and 18 days). IDH1 R132 or IDH2 R172 hotspot mutations were not detected in any of the samples. The absence of IDH1 or IDH2 mutations in paediatric MB may be attributed to differences in mutational profiles and cellular origins in childhood MB, despite its histomolecular similarities with adult MB.
Osteoarthritis (OA) is a degenerative arthritis associated with aging. It is recognized that telomere attrition is a hallmark of aging. However, the transcriptional dynamics of synovial telomere-related genes (TRGs) in OA has not yet been elucidated. OA synovium microarray profiles were sourced from GEO and TRGs from TelNet. GO, KEGG, DO and GSVA enrichment analyses were employed to explore the underlying mechanisms. WGCNA and machine learning methods were utilized to screen hub differentially expressed TRGs (TRDEGs) that highly correlated with OA traits (hub OA-TRDEGs). Nomograms and receiver operating characteristic (ROC) curves were used to evaluate the diagnostic performance of hub OA-TRDEGs. An RNA-binding protein (RBP) network was developed to predict potential RBP target genes.​ The CIBERSORT analysis was performed to assess associations between hub OA-TRDEGs and immune infiltration. We identified 77 TRDEGs in normal and OA synovium samples. Functional enrichment analysis implicated these ge-nes primarily in metabolism regulation, DNA repair and inflammatory response. LTA4H, HNMT, ANKMY2, UFSP2, HLTF and RPA3 were established as hub OA-TRDEGs, demonstrating strong diagnostic performance for OA. Wilcox testing confirmed significant up-regulation of all hub OA-TRDEGs in OA synovium - a finding validated through independent datasets and qRT-PCR assays. Immune infiltration analysis further indicated that ​​resting mast cells, CD4+ memory resting T cells and activated mast cells are implicated in OA pathogenesis and exhibit significant correlations with hub OA-TRDEGs. These results nominate hub OA-TRDEGs as potential dia-gnostic biomarkers and underscore immune cell infiltration as a critical driver of OA progression.
Trypsin (TRY) combined with ethylenediaminetetraacetic acid (EDTA) is a widely used dissociation agent due to its efficiency and cost-effectiveness. However, its impact on preserving stem cell marker expression, such as C-X-C chemokine receptor type 4 (CXCR4) (critical for cell migration and homing) and cluster of differentiation 146 (CD146) (involved in pluripotency and angiogenesis), may be suboptimal compared to alternatives such as Accuta-se (ACC) and Accumax (ACMX), as shown previous-ly in bone marrow-derived stem cells (BM-MSCs). Limited data exist on these agents' effects on dental pulp stem cells (DPSCs). This study aims to investigate the influence of TRY, ACC, and ACMX on the expression of CXCR4 and CD146 in DPSCs. Seven characterized DPSC lines were cultured under standardized conditions and detached using TRY-EDTA, ACC, or ACMX. The expression of CXCR4 and CD146 was quantified via multicolour flow cytometry using an innovative DURAClone SC panel with supplementary anti-CXCR4 antibody. Comprehen-sive statistical analyses were performed to evaluate differences in marker preservation. No statistically significant differences in CXCR4 or CD146 expression were observed across the detachment methods (P > 0.05). ACMX consistently demonstrated margi-nally higher mean expression levels for both markers (CXCR4: 84.77 %; CD146: 93.91 %) compared to ACC (CXCR4: 83.45 %; CD146: 93.41 %) and TRY (CXCR4: 83.95 %; CD146: 92.99 %). While differences were not statistically significant, ACMX consistently yielded higher mean expression of both CXCR4 and CD146, indicating a potential advantage in preserving marker integrity during the cell detachment.
Chronic obstructive pulmonary disease (COPD) is a leading cause of global mortality, characterized by persistent airflow limitation and chronic inflammation. Emerging evidence suggests that long non-coding RNAs (lncRNAs) play regulatory roles in COPD pathogenesis, but the function of DPP10-AS1 remains unclear. This study aimed to investigate the expression pattern, diagnostic value and functional mechanism of DPP10-AS1 in COPD, particularly its role in cigarette smoke-induced airway epithelial injury in 16HBE cells. Bioinformatic analysis of the GSE201465 dataset identified DPP10-AS1 as significantly up-regulated in COPD. Clinical validation was performed in 80 COPD patients and 80 controls. In vitro, 16HBE cells were treated with cigarette smoke extract (CSE) following DPP10-AS1 knockdown/over-expression. The interaction between DPP10-AS1 and miR-34c-5p was verified through RNA pull-down and dual-luciferase reporter assays. Functional rescue experiments with miR-34c-5p inhibitor in DPP10-AS1-silenced cells under CSE exposure were constructed. DPP10-AS1 expression was markedly elevated in COPD patients, showing a significant correlation with impaired lung function (FEV1/FVC: r = -0.743). It presented a diagnostic potential (AUC = 0.806). CSE exposure up-regulated DPP10-AS1 in 16HBE cells, which exacerbated inflammation (IL-6, IL-8 and COX-2) and fibrosis (α-SMA, Col1A1 and TGF-β1). Our research results indicated that DPP10-AS1 acts as a "molecular sponge" for miR-34c-5p. This mecha-nism may be of great significance for its role in COPD. We concluded that DPP10-AS1 promotes CSE-induced airway inflammation and remodelling through miR-34c-5p in 16HBE cells, suggesting its potential involvement in COPD progression. These findings combined with clinical analysis position DPP10-AS1 as a candidate diagnostic biomarker and therapeutic target.
Fragility fractures have been a cause for concern because of their high incidence. For the prevention and treatment of osteoporotic fractures, it is important to understand how to promote bone formation and increase bone mass. This study investigated miR-296-3p expression and function in fragility fracture. The study enrolled 98 patients with hip fractures, 90 patients with wrist fractures and 35 healthy controls. RT-qPCR was used to detect the miR-296-3p level changes before and after surgery in fracture patients and during the differentiation of human bone mesenchymal stem cells (BMSCs). The starBase bioinformatics database was used for prediction of the miR-296-3p target gene, and dual luciferase report was used for verification of the target relationship. Our results demonstrated that miR-296-3p levels are up-regulated in fracture patients, while they gradually decrease during human BMSC differentiation. The up-regulation of miR-296-3p inhibited the proliferation and differentiation ability of human BMSCs, while inhibition of its expression had the opposite effects. miR-296-3p negatively regulates osteogenic differentiation, and over-expression of inhibitor of β-catenin and TCF (ICAT) could counteract the negative regulatory effect. miR-296-3p targets ICAT and affects the expression of key proteins in the Wnt/β-catenin signalling pathway. In conclusion, miR-296-3p can regulate the division and differentiation of osteoblasts by affecting the expression of ICAT and participate in fracture healing.
Intracerebral haemorrhage (ICH) is a severe stroke subtype with high mortality and poor functional recovery. Neuroinflammation, mediated by NLRP3 inflammasome and caspase-1 (Casp1) activation, drives secondary brain injury, including oedema and cell death after ICH. We hypothesize that combining NLRP3 inhibitor OLT1177 and Casp1 inhibitor VX765 will provide enhanced neuroprotection against brain oedema and injury in experimental ICH. ICH was induced by injecting autologous blood into the basal ganglia in mouse models. Sixty-three C57Bl/6 male mice were randomly assigned to five groups: sham, vehicle, OLT1177 (dapansutrile, 200 mg/kg intraperitoneally), VX765 (75 mg/kg intraperitoneally) and combination of OLT1177 and VX765. Mice were treated for three consecutive days, starting 1 hour after ICH surgery. Behavioural tests, brain oedema, brain water content, blood-brain barrier integrity, vascular permeability, cell apoptosis, and levels of NLRP3 and its downstream proteins were measured. OLT1177 significantly reduced cerebral oedema after ICH and improved neurological function. It preserved blood-brain barrier integrity and reduced vascular leakage. Furthermore, OLT1177 prevented micro-glial morphological changes and significantly inhibited activation of Casp1 and release of IL-1β. OLT1177 also protected against neuronal loss in the affected hemisphere. Notably, the combination of OLT1177 and VX765 further attenuated brain injury after ICH by inhibiting inflammasome activation. The combination of OLT1177 and VX765 thus provided enhanced protection against brain injury by inhibiting inflammasome activation, suggesting that OLT1177 in combination with VX765 might serve as a promising therapeutic strategy for the treatment of ICH.
Long noncoding RNAs (lncRNAs) are known to play critical roles in the progression of osteosarcoma. Despite their recognized importance, the specific biological functions of lncRNAs in osteosarcoma remain unclear. In this context, prostate cancer-associated transcript 7 (PCAT7) has been identified as a bone metastasis-related lncRNA through the analysis of The Cancer Genome Atlas dataset. In this study, we investigated the expression of PCAT7 in osteosarcoma cells, particularly those exhibiting resistance to doxorubicin, a widely used chemotherapeutic agent in clinic. Functional assays including cell growth, invasion and apoptosis were conducted to elucidate the impact of PCAT7 inhibition on osteosarcoma cells, focusing on sensitivity to doxorubicin treatment. To understand the underlying molecular mechanisms, the interaction between PCAT7, miR-324-5p, and the TGF-β/SMAD signalling pathway was further explored. The study revealed that PCAT7 is up-regulated in osteosarcoma cells with doxorubicin resistance. Inhibition of PCAT7 could enhance the sensitivity to doxorubicin treatment by reducing cell growth, suppressing cell invasion and increasing cell apoptosis. Mechanistically, PCAT7 was shown to activate the TGF-β/SMAD signalling pathway by up-regulating the expression of TGFBR1 through sponging miR-324-5p. These findings unveil a novel mechanism contributing to the constitutive activation of TGF-β signalling in osteosarcoma. Targeting PCAT7 may offer a promising avenue for therapeutic interventions in osteosarcoma by disrupting the aberrant TGF-β signalling, thus presenting a potential strategy to improve treatment outcomes in this challenging cancer.
The aim of this study was to analyse the allelic distribution of selected genes in the Czech and Vietnamese populations. We analysed samples from 94 Vietnamese volunteers and 2,859 Czech population-based subjects (2,559 from the Czechs post-MONICA and 300 volunteers from the South region of the Czech Republic). There were significant differences between the two populations for most, but not all, of the SNPs analysed. In particular, the prevalence of risk alleles in the analysed polymorphisms tended to be lower in the Vietnamese community compared to the Czech population, especially within the FTO (rs17817449; associated with obesity risk, P < 0.0001), TCF7L2 (rs7903146; linked to type 2 dia-betes, P < 0.0001) and ADH1B (rs1229984; related to alcohol consumption, P < 0.0001) genes. The genotype within the MCM6/LCT cluster (rs4988235) associated with lactase persistence was not present in the Vietnamese population. Slight genotype differences were detected for one HFE polymorphism (rs1799945 with P = 0.005; but not for rs1800562). Only the genotype frequencies within the MC4R and APOE genes were almost identical in both populations. We conclude that the Vietnamese population may have a lower genetic predisposition to the non-communicable diseases such as obesity or diabetes mellitus.
Statins are the most commonly prescribed lipid-lowering drugs. Around 10 % of statin users develop one of statin-associated muscle syndromes (SAMS), ranging from mild myalgia to severe life-threatening rhabdomyolysis. So far, no reliable marker of SAMS has been identified. Plasma cell-free DNA (cfDNA, of nuclear or mitochondrial origin) and microRNA (miRNA) are among the potential molecules that could reflect muscle changes induced by statins. Two groups of statin-treated subjects were included into our study. In an intensively screened branch, 15 males on 10 mg of rosuvastatin provided two samples before the treatment initiation, three subsequent at weeks 4, 8 and 12 on statin treatment, and one after four weeks of the wash-out period. The second branch included 29 subjects, with samples collected before the treatment and after 5-6 and 10-12 months of treatment. Plasma cfDNA of nuclear or mitochondrial origin and miRNAs 133a-3p, 23a-5p and 1-3p were analysed using qPCR. Plasma concentrations of cfnDNA, cfmtDNA or miRNAs did not change significantly during the statin treatment (all P values between 0.27-0.93). The release of cfDNA and miRNA is probably not significantly affected by low-dose statin therapy.
With a dismal prognosis, cholangiocarcinoma (CCA) is a highly invasive cancer and its global incidence is increasing. Non-coding RNAs, particularly circRNAs, are increasingly recognized as important regulators in tumorigenesis, yet the mechanistic details of hsa_circ_0000977-mediated miR-338-3p regulation in CCA remain incompletely understood. This study aimed to investigate the mechanism of the hsa_circ_0000977/miR-338-3p/ETS1 axis in CCA progression, evaluate its prognostic significance and investigate its functional role in CCA cells. Real-time quantitative PCR (RT-qPCR) was performed to quantify hsa_circ_0000977, miR-338-3p and ETS1 mRNA expression levels. Pearson correlation was used to assess their association. To confirm the molecular interactions between these molecules, dual-luciferase reporter assays were employed. Cell migration capacity was evaluated via Transwell migration assays, while CCK-8 tests analysed proliferation. Prognostic value was assessed through survival analysis and multi-variate regression. The study revealed significant up-regulation of hsa_circ_0000977 in CCA tumour tissues, with elevated expression levels associated with poorer 5-year survival outcomes. Multivariate analysis confirmed that hsa_circ_0000977 over-expression is an independent predictor of survival. Functional assays indicated that hsa_circ_0000977 negatively regulated miR-338-3p, which was down-regulated in CCA and exhibited tumour-suppressive effects. CCA cell proliferation and migration were suppressed following hsa_circ_0000977 knockdown, effects that were partially reversed by miR-338-3p inhibition. Further investigation demonstrated that miR-338-3p exerts its tumour-suppressive effects by directly targeting ETS1, and the hsa_circ_0000977/miR-338-3p/ETS1 axis regulates the proliferation and migration of CCA cells. We have concluded that hsa_circ_0000977 drives CCA progression by sponging miR-338-3p and modulating its target ETS1, suggesting its clinical value for predicting outcomes and developing targeted therapies.
Ovarian cancer remains one of the most lethal gynaecological malignancies, with paclitaxel resistance being a major therapeutic challenge that limits treatment efficacy and patient survival. We found that although the BARD1 level was not signi-ficantly altered in patients with ovarian cancer (OC), patients with higher BARD1 levels had increased survival time, suggesting that the down-regulation of BARD1 may be related to the paclitaxel sensitivity. Through examining the expression of BARD1 in tumour samples from paclitaxel responders and non-responders, we observed that the BARD1 level was significantly reduced in non-responders. CYP2C8 was up-regulated in non-responders. Also, the BARD1 level was negatively correlated with the level of CYP2C8. BARD1 over-expression in OC cells could repress the CYP2C8 expression, while knockdown of BARD1 could up-regulate CYP2C8 expression, which could be rescued by H2A-Ub. Results from gain and loss of functional experiments indicated that BARD1 functions as a tumour suppressor during paclitaxel treatment, and BARD1 down-regulation increased the IC50 of paclitaxel from 2.46 nM to 5.33 nM in SK-OV-3 cells and from 3.11 nM to 7.51 nM in CaoV-3 cells. We are the first to demonstrate that the down-regulation of BARD1 contributes to paclitaxel resistance via up-regulating CYP2C8 in patients with OC, which provides a potent target for clinical OC treatment.
High-throughput, precise and cost-effective isolation of high-quality RNA is essential for the growing number of RNA-based next-generation sequencing (NGS) analyses. Manual RNA isolation provides sufficient quality but requires significant hands-on time and carries an increased risk of contamination and sample misidentification. Here we describe a semi-automated protocol for the isolation of high-quality total RNA from 3 ml of peripheral blood collected in Tempus Blood RNA Tubes. The isolation can be performed either from the total volume of 9 ml of Tempus blood lysate or from smaller volumes (6 and 3 ml, respectively) using the MagCore triXact RNA Kit on the MagCore Plus II automated nucleic acid extractor, which allows RNA isolation in single tubes. The original isolation protocol (#631) for whole blood RNA isolation was customized by the manufacturer (#631T) by omitting the cell lysis step. After optimizing the process, we compared the yield and quality of 760 RNA samples isolated manually or by semi-automated methods. We conclude that RNA isolation using the semi-automated MagCore protocol yields 5-10 μg of total RNA from 6 ml of lysate (2 ml of peripheral blood), which is almost comparable in quantity and quality to manual isolation. In addition, we show that the remaining 3 ml of lysate is sufficient for backup re-isolation. Our semi-automated RNA protocol reduces hands-on time without increasing costs and yields bulky total RNA of a quali-ty suitable for subsequent RNA NGS applications.