RNA Purity Research Articles

PKA and NBC expression patterns in cryopreserved bovine spermatozoa after kaempferol treatment

Kaempferol (KAE) is a member of the flavonoid family, which exhibits antioxidant properties. The richest sources of KAE are green leafy vegetables and herbs like kale, spinach or dill. The goal of our study was to evaluate a possible cryoprotective effects of KAE through to gene expression of protein kinase A (PKA) and sodium bicarbonate cotransporter (NBC). For the experiments we used semen samples obtained from 12 adult Holstein bulls. Before cryopreservation, all samples were divided equally and supplemented by different concentrations of KAE (0, 12.5, 25 and 50 µmol/L), except for native control. After that, RNA was isolated, and the purity and quality of RNA was verified nanophotometrically at A260/A280 nm. Next step was a transcription of RNA into cDNA, which was used for two-step qPCR while expression of PKA and NBC was quantified under specific cycling conditions. Data was evaluated by the delta delta Ct method and One-way ANOVA using Tukey’s range test. According to expression patterns, it was observed that 25 µmol/L of KAE significantly preserved (P<0.0001) the PKA compared to cryopreserved control without KAE treatment. In the case of NBC, there was slightly increase of expression in the group treated with 50 µmol/L of KAE but without significant changes. In conclusion, KAE treatment exhibit promising cryoprotective properties of PKA and NBC genes especially in higher concentration 25 and 50 µmol/L, which may successfully preserved proper expression after thawing of cryopreserved bovine spermatozoa.

Selection of optimal extraction and RT-PCR protocols for stool RNA detection of colorectal cancer associated immune genes

There is a growing interest in using fecal mRNA transcripts as biomarkers for non-invasive detection of colorectal cancer (CRC). The following study compares different RNA extraction and reverse transcription PCR (RT-PCR) methods for mRNA detection in stool and identifies a robust and sensitive protocol. A combination of the Stool total RNA purification kit (Norgen) and the Superscript III one-step RT-PCR kit (Invitrogen) provided high RNA purity and sensitive and consistent mRNA detection, making them well-suited candidates for large-scale studies. We tested the protocol by detecting the mRNA of several immune genes (CXCL1, IL8, IL1B, IL6, PTGS2, and SPP1) in 22 CRCs, 24 adenomatous polyps, and 22 control stool samples. All these inflammatory markers, except for CXCL1, showed a strong association with CRC. Cancer stool samples showed increased levels of IL1B, IL8, and PTGS2 transcripts compared to polyp and control groups. Thus, this work supports the potential use of fecal mRNA as biomarkers for CRC detection.

Expression of Plasma miRNA-133a is Significantly Lower in Acute Coronary Syndrome (ACS) than in Healthy/Non-ACS Subjects

BACKGROUND: The current biomarker diagnostic modality for acute coronary syndrome (ACS), cardiac troponin, has several limitations. Emerging studies showed that micro-RNA (miR)-133a was released from infarcted heart to circulation, yet the diagnostic value of miR-133a in ACS demonstrated a conflicting result. Therefore, this study was conducted to investigate the potency of plasma miR-133a as a biomarker candidate of ACS.METHODS: This was a case-controlled study, involving ACS and control subjects. The sociodemographic and clinical characteristics were assessed through medical records. A final of 39 ACS and 31 control subjects (consist of healthy and non-ACS subjects) passed the selection procedure by demonstrating a high purity of RNA. miR-133a from ACS and control subjects were detected by quantitative polymerase chain reaction (qPCR). Expression of miR-133a was evaluated for sensitivity and specificity as an ACS biomarker diagnostic using the receiver operating characteristic (ROC) curve.RESULTS: Plasma miR-133a expression was stably found in ACS subjects. The plasma miR-133a level was lower in ACS than in control subjects. miR-133a effectively distinguished ACS subjects from healthy subjects (AUC=0.911) and exhibited high diagnostic performance, with a sensitivity of 87.1% and specificity of 100% at a cut-off value of 44.035. In an extended model including both control subjects (healthy and non-ACS with comorbid conditions), miR-133a maintained diagnostic significance (AUC=0.874), showing sensitivity of 76.9% and specificity of 100% at a cut-off value of 11.69.CONCLUSION: Plasma miR-133a is significantly lower and effectively distinguishes ACS patients from both healthy individuals and non-ACS individuals with comorbid, with a cut-off value of 11.69. Therefore, plasma miR-133a is suggested to be a good candidate for diagnostic biomarkers of ACS.KEYWORDS: circulating miRNA, miRNA-133a, acute coronary syndrome, diagnostic biomarker

Comparative investigation of exosome extraction from rat bone marrow mesenchymal stem cells using three different methodologies

AbstractExosomes have been identified as crucial mediators in numerous physiological and pathological processes, emerging as a focal point of scientific inquiry. This study aims to compare three methods for isolating exosomes from rat bone marrow mesenchymal stem cells: ultracentrifugation (UC), ultrafast separation system (EXODUS), and commercial precipitation kit (EXO‐kit). First, the investigation compared exosomal morphology, particle size distribution, and expression of marker proteins. Subsequently, the RNA content, protein concentration, and purity of exosomes were evaluated. Finally, the impact of these exosomes on cellular metabolic viability and migration capacity was assessed. Results indicated that exosomes exhibited spherical or elliptical membrane structures, and most of the exosomes extracted by the three methods were in the range of 30to 200 nm. UC‐extracted exosomes demonstrated the least impurities and clearest background, followed by EXODUS‐extracted exosomes, and lastly EXO‐kit‐extracted exosomes. The EXO‐kit‐extracted exosomes yielded the highest RNA and protein content, whereas those isolated through UC exhibited superior purity. Furthermore, exosomes extracted from EXODUS and EXO‐kit methods effectively enhanced the metabolic viability and migratory ability of osteoblast precursor cells compared to UC‐extracted exosomes. In conclusion, each of the three methodologies presents advantages and limitations. Therefore, the selection of an appropriate exosome extraction technique should be based on specific experimental objectives and requirements.

Evaluation of commercial RNA extraction kits for long-read metatranscriptomics in soil

Metatranscriptomic analysis of the soil microbiome has the potential to reveal molecular mechanisms that drive soil processes regulated by the microbial community. Therefore, RNA samples must be of sufficient yield and quality to robustly quantify differential gene expression. While short-read sequencing technology is often favoured for metatranscriptomics, long-read sequencing has the potential to provide several benefits over short-read technologies. The ability to resolve complete transcripts on a portable sequencing platform for a relatively low capital expenditure makes Oxford Nanopore Technology an attractive prospect for addressing many of the challenges of soil metatranscriptomics. To fully enable long-read metatranscriptomic analysis of the functional molecular pathways expressed in these diverse habitats, RNA purification methods from soil must be optimised for long-read sequencing. Here we compare RNA samples purified using five commercially available extraction kits designed for use with soil. We found that the Qiagen RNeasy PowerSoil Total RNA Kit performed the best across RNA yield, quality and purity and was robust across different soil types. We found that sufficient sequencing depth can be achieved to characterise the active community for total RNA samples using Oxford Nanopore Technology, and discuss its current limitations for differential gene expression analysis in soil studies.

Development and validation of a rapid five-minute nucleic acid extraction method for respiratory viruses

BackgroundThe rapid transmission and high pathogenicity of respiratory viruses significantly impact the health of both children and adults. Extracting and detecting their nucleic acid is crucial for disease prevention and treatment strategies. However, current extraction methods are laborious and time-consuming and show significant variations in nucleic acid content and purity among different kits, affecting detection sensitivity and efficiency. Our aim is to develop a novel method that reduces extraction time, simplifies operational steps, and ensures high-quality acquisition of respiratory viral nucleic acid.MethodsWe extracted respiratory syncytial virus (RSV) nucleic acid using reagents with different components and analyzed cycle threshold (Ct) values via quantitative real-time polymerase chain reaction (qRT-PCR) to optimize and validate the novel lysis and washing solution. The performance of this method was compared against magnetic bead, spin column, and precipitation methods for extracting nucleic acid from various respiratory viruses. The clinical utility of this method was confirmed by comparing it to the standard magnetic bead method for extracting clinical specimens of influenza A virus (IAV).ResultsThe solution, composed of equal parts glycerin and ethanol (50% each), offers an innovative washing approach that achieved comparable efficacy to conventional methods in a single abbreviated cycle. When combined with our A Plus lysis solution, our novel five-minute nucleic acid extraction (FME) method for respiratory viruses yielded superior RNA concentrations and purity compared to traditional methods. FME, when used with a universal automatic nucleic acid extractor, demonstrated similar efficiency as various conventional methods in analyzing diverse concentrations of respiratory viruses. In detecting respiratory specimens from 525 patients suspected of IAV infection, the FME method showed an equivalent detection rate to the standard magnetic bead method, with a total coincidence rate of 95.43% and a kappa statistic of 0.901 (P < 0.001).ConclusionsThe FME developed in this study enables the rapid and efficient extraction of nucleic acid from respiratory samples, laying a crucial foundation for the implementation of expedited molecular diagnosis.

Association of XRCC2 with breast cancer, a multi-omics analysis at genomic, transcriptomic, and epigenomic level.

One of the main causes of cancer-related mortality for women worldwide is breast cancer (BC). The XRCC2 gene, essential for DNA repair, has been implicated in cancer susceptibility. This study aims to evaluate the association between XRCC2 and BC risk. The study was conducted at Zheen International Hospital in Erbil, Iraq, between 2021 and 2024 with a total of 88 samples, including 44 paired normal and cancer tissue samples. Mutation analysis was performed using Next-Generation Sequencing, coupled with in silico tools for variant impact prediction. Expression levels were assessed through RT-PCR, and methylation status was determined using methylation-sensitive restriction enzyme digestion PCR. The study identified seven inherited germline variants in the XRCC2 gene, with five of these mutations being Uncertain Significance, one being Likely Pathogenic, and one being Likely benign. RNA purity was found high with mean A260/280 ratios of 1.986 ± 0.097 in normal (N) and 1.963 ± 0.092 in tumor (T) samples. Tumor samples exhibited a higher RNA concentration (78.56 ± 40.87 ng/µL) than normal samples (71.44 ± 40.79 ng/µL). XRCC2 gene expression was significantly upregulated in tumor tissue, with marked increases in patients aged 40-55 and >56 years and in higher cancer grades (II and III) and invasive ductal carcinoma (p-values ranging from <0.0001 to 0.0392). DNA methylation rates in tumor tissues were low (7%), suggesting limited regulation by methylation. The study suggests that XRCC2 can be classified as an oncogene and that its structural investigation by targeted NGS and expression evaluation can be used as a potential biomarker in BC.

Evaluation of Multiple RNA Extraction Protocols for Chikungunya Virus Screening in Aedes aegypti Mosquitoes.

Chikungunya virus (Togaviridae, Alphavirus; CHIKV) is a mosquito-borne global health threat. The main urban vector of CHIKV is the Aedes aegypti mosquito, which is found throughout Brazil. Therefore, it is important to carry out laboratory tests to assist in the virus's diagnosis and surveillance. Most molecular biology methodologies use nucleic acid extraction as the first step and require quality RNA for their execution. In this context, four RNA extraction protocols were evaluated in Ae. aegypti experimentally infected with CHIKV. Six pools were tested in triplicates (n = 18), each containing 1, 5, 10, 20, 30, or 40 mosquitoes per pool (72 tests). Four commercial kits were compared: QIAamp®, Maxwell®, PureLink®, and PureLink® with TRIzol®. The QIAamp® and PureLink® with TRIzol® kits had greater sensitivity. Two negative correlations were observed: as the number of mosquitoes per pool increases, the Ct value decreases, with a higher viral load. Significant differences were found when comparing the purity and concentration of RNA. The QIAamp® protocol performed better when it came to lower Ct values and higher RNA purity and concentration. These results may provide help in CHIKV entomovirological surveillance planning.

Fine-needle aspiration technique under endoscopic ultrasound guidance: A technical approach for RNA profiling of pancreatic neoplasms.

Early diagnosis of pancreatic ductal adenocarcinoma (PDAC) has been a longstanding challenge. The prognosis of patients with PDAC depends on the stage at diagnosis. It is necessary to identify biomarkers for the detection and differentiation of pancreatic tumors and optimize PDAC sample preparation procedures for DNA and RNA analysis. Most molecular studies are done using paraffin-embedded blocks; however, the integrity of DNA and RNA is often compromised in this format. Moreover, RNA isolated from human pancreatic tissue samples is generally of low quality, in part, because of the high concentration of endogenous pancreatic RNAse activity present. To assess the potential of endoscopic ultrasound-guided fine-needle aspiration (EUS-FNA) to obtain specimens from pancreatic neoplasms for subsequent RNA molecular profiling, including next-generation sequencing (NGS). Thirty-four EUS-FNA samples were included in this study: PDAC (n = 15), chronic pancreatitis (n = 5), pancreatic cysts (n = 14), mucinous cysts (mucinous cystic neoplasia/intraductal papillary mucinous neoplasia) n = 7, serous cystic neoplasms n = 5, and pseudocysts n = 2. Cyst material consisted of cyst fluid and cyst wall samples obtained by through-the-needle biopsy (TTNB). Samples were stored at -80 °C until analysis. RNA purity (A260/230, A260/280 ratios), concentration, and integrity (RIN) were assessed. Real-time polymerase chain reaction was conducted on all samples, and small RNA libraries were prepared from solid mass samples. RNA was successfully extracted from 29/34 (85%) EUS-FNA samples: 100% pancreatic adenocarcinoma samples, 100% chronic pancreatitis samples, 70% pancreatic fluid cyst samples, and 50% TTNB samples. The relative expression of GAPDH and HPRT were obtained for all successfully extracted RNA samples (n = 29) including low-quality RNA specimens. Low concentration and nonoptimal RIN values (no less than 3) of RNA extracted from EUS-FNA samples did not prevent NGS library preparation. The suitability of cyst fluid samples for RNA profiling varied. The quality of RNA extracted from mucinous cyst fluid had a median RIN of 7.7 (5.0-8.2), which was compatible with that from solid neoplasms [6.2 (0-7.8)], whereas the quality of the RNA extracted from all fluids of serous cystic neoplasms and TTNB samples had a RIN of 0. The results demonstrate the high potential of EUS-FNA material for RNA profiling of various pancreatic lesions, including low-quality RNA specimens.

Contact lenses as novel tear fluid sampling vehicles for total RNA isolation, precipitation, and amplification.

The tear fluid is a readily accessible, potential source for biomarkers of disease and could be used to monitor the ocular response to contact lens (CL) wear or ophthalmic pathologies treated by therapeutic CLs. However, the tear fluid remains largely unexplored as a biomarker source for RNA-based molecular analyses. Using a rabbit model, this study sought to determine whether RNA could be collected from commercial CLs and whether the duration of CL wear would impact RNA recovery. The results were referenced to standardized strips of filtered paper (e.g., Shirmer Strips) placed in the inferior fornix. By performing total RNA isolation, precipitation, and amplification with commercial kits and RT-PCR methods, CLs were found to have no significant differences in RNA concentration and purity compared to Schirmer Strips. The study also identified genes that could be used to normalize RNA levels between tear samples. Of the potential control genes or housekeeping genes, GAPDH was the most stable. This study, which to our knowledge has never been done before, provides a methodology for the detection of RNA and gene expression changes from tear fluid that could be used to monitor or study eye diseases.

Sampling methodology and storage management effect on deformed wing virus (DWV) quantification using real-time PCR

Deformed wing virus (DWV) is one of the most important viruses affecting Apis mellifera. A valid method for estimating the viral load at the honey bee colony level is of utmost importance. In this study, different samplings of bees were performed, in order to determine the appropriate sample size and collection point in the hive. A similar trend was observed at both sampling points, and subsequently, the DWV load was quantified in samples collected from the entire hive area. Whether 10 or 50 bees were sampled from each frame, the viral titre showed a similar dispersion and the lowest coefficient of variance was recorded in 10 bees/frame samples. According to our results, each one of the tested sampling methods can be used to quantify the viral load at the colony level, eliminating the need to sample 50 bees per frame due to the large number of bees. The effect of post-collection sample management, specifically the combination of storage temperature and duration, on RNA extraction yield, purity and virus quantification results was also investigated. The DWV titre determination was successful for all groups. It seems that improper storage affects the results of the viral quantification, however, significant differences were not observed among the treatments. Undoubtedly, immediate sample preservation in extremely low temperatures is the most appropriate method for virus analyses.

Comparison of Fecal MicroRNA Isolation Using Various Total RNA Isolation Kits.

Fecal specimens have long been regarded as promising sources for gastrointestinal cancer screening and have, thus, been extensively investigated in biomarker research. MicroRNAs (miRNAs) are small, non-coding RNA molecules involved in regulating various biological processes. They are commonly dysregulated during tumor development and exhibit differential expression in feces. To assess the preanalytical feasibility of fecal miRNA analysis, we systematically compared the performance of commonly used total RNA extraction methods. Fecal samples from healthy subjects were utilized for this evaluation. Various methods, including miRNeasy, Universal, Trizol, RNeasy, and mirVana kits, were employed to isolate total RNA. MiRNA expression analyses were conducted using TaqMan or SYBR Green qRT-PCR for a subset of miRNAs, with externally spiked-in cel-miR-39 used for normalization. Most methods demonstrated similar performance in terms of the total RNA concentration and purity. Externally spiked cel-miR-39 and endogenous miRNAs (RNU6b, miR-16, and miR-21) exhibited comparable concentrations across the different RNA isolation methods, whereas the RNeasy mini kit consistently yielded lower values. Our findings suggest that various isolation methods produce reproducible and comparable miRNA expression results, supporting the potential comparability and translational applicability of miRNA-based biomarker research in the future.

Principles and Limitations of miRNA Purification and Analysis in Whole Blood Collected during Ablation Procedure from Patients with Atrial Fibrillation.

Background: MicroRNA (miRNA) have the potential to be non-invasive and attractive biomarkers for a vast number of diseases and clinical conditions; however, a reliable analysis of miRNA expression in blood samples meets a number of methodological challenges. In this report, we presented and discussed, specifically, the principles and limitations of miRNA purification and analysis in blood plasma samples collected from the left atrium during an ablation procedure on patients with atrial fibrillation (AF). Materials and Methods: Consecutive patients hospitalized in the First Department of Cardiology for pulmonary vein ablation were included in this study (11 with diagnosed paroxysmal AF, 14 with persistent AF, and 5 without AF hospitalized for left-sided WPW ablation-control group). Whole blood samples were collected from the left atrium after transseptal puncture during the ablation procedure of AF patients. Analysis of the set of miRNA molecules was performed in blood plasma samples using the MIHS-113ZF-12 kit and miScript microRNA PCR Array Human Cardiovascular Disease. Results: The miRNS concentrations were in the following ranges: paroxysmal AF: 7-23.1 ng/µL; persistent AF: 4.9-66.8 ng/µL; controls: 6.3-10.6 ng/µL. The low A260/280 ratio indicated the protein contamination and the low A260/A230 absorbance ratio suggested the contamination by hydrocarbons. Spectrophotometric measurements also indicated low concentration of nucleic acids (<10 ng/µL). Further steps of analysis revealed that the concentration of cDNA after the Real-Time PCR (using the PAXgene RNA Blood kit) reaction was higher (148.8 ng/µL vs. 68.4 ng/µL) and the obtained absorbance ratios (A260/A280 = 2.24 and A260/A230 = 2.23) indicated adequate RNA purity. Conclusions: Although developments in miRNA sequencing and isolation technology have improved, detection of plasma-based miRNA, low RNA content, and sequencing bias introduced during library preparation remain challenging in patients with AF. The measurement of the quantity and quality of the RNA obtained is crucial for the interpretation of an efficient RNA isolation.

Abstract 4944: Divergent proteogenomic gene expression is driven by microenvironment across tumor types

Abstract Solid tumors develop through a complex series of genome and downstream expression alterations that interact with the local tissue microenvironment, leading to a transformed cell and malignant phenotype. With massive multi-omic data, recent proteogenomics studies have jointly analyzed RNA and protein expression to uncover new gene regulatory relationships; however, many of these studies focused on single tumor types and lack a generalizable view of joint RNA and protein expression. Here, we present the first pan-cancer analysis of sample-wise RNA to protein correlation (SRPC). We re-analyzed over 1,000 tumors across 10 tumor types from published proteogenomic data from the Clinical Proteomic Tumor Analysis Consortium (CPTAC) and the Applied Proteogenomics OrganizationaL Learning and Outcomes (APOLLO) Research Network. We identified a wide range of SRPC across all tumors (ρ range: -0.08 to 0.70, median: 0.45). We then analyzed tumor pathologic and molecular characteristics versus the range of SRPC. High SRPC tumors had high tumor purity by pathology review, by somatic DNA whole exome sequencing, and by RNA and protein purity scores. In contrast, low SRPC tumors had high immune cell and stromal cell expression scores as inferred by either protein or RNA based signatures. We observed marked differences in cell type populations estimated by expression in different SRPC tumor tranches (low SRPC: macrophages, endothelial cells, cancer-associated fibroblasts; high SRPC: CD4+ Th2 cells). Tumors expressed signaling pathways depending on their SRPC (low SRPC: hypoxia, inflammatory response, and KRAS signaling; high SRPC: DNA repair and E2F targets). SRPC also stratifies tumors with distinct somatic DNA alterations (low SRPC: HRAS and NF2; high SRPC: TP53, MEN1, high Tumor Mutational Burden and high DNA chromosomal instability). Finally, we found that cancer driver genes displayed divergent gene-wise RNA to protein correlations (GRPC) among tumor types with a median absolute deviation interquartile range of 0.1 - 0.2, suggesting tumor-type-specific regulation. In summary, divergent RNA and protein expression is driven, in part, by tumor microenvironment composition across tumor types. Tumors with a diverse cellular microenvironment display a summation of RNA and protein expression resulting from this cell type diversity leading to a low SRPC, while tumors predominated by tumor cells display coordinated RNA and protein expression levels resulting from a pure clonal or cell type leading to a high SRPC. This first deep analysis into sample-wise RNA to protein correlation represents a large proteogenomic community resource for informing biomarker analysis by modality and by tranches of tumor microenvironment. The views expressed in this abstract are solely of the authors and do not reflect the official policy of the Departments of Army/Navy/Air Force, Department of Defense, USUHS, HJF, or U.S. Government. Citation Format: Joseph LaMorte, Nicholas Bateman, Thomas Conrads, Robert F. Browning, Craig D. Shriver, Robert L. Kortum, Matthew D. Wilkerson. Divergent proteogenomic gene expression is driven by microenvironment across tumor types [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4944.

RNA isolation by two different methods in analysing adipose-biomarkers of mouse adipose tissues

The RNeasy Mini Kit and the TRI Reagent protocol are commonly employed techniques for extracting total RNA and are widely utilised in research pertaining to the expression of adipose biomarkers. This study was undertaken to establish and determine an appropriate method for isolating RNA from frozen adipose tissue, with the objective of analysing adipose biomarkers. Total RNA was extracted from frozen white adipose tissues of mice (-80oC) using both the RNeasy Mini Kit and the TRI Reagent protocol. The concentration and purity of the RNA were assessed using NanoDrop. One-step RT-qPCR was employed to quantify the mRNA expression levels of three adipose biomarkers: MEST, SFRP5, and SCD1. The results of this investigation indicated that the RNeasy Mini Kit yielded a higher RNA concentration when compared to the TRI Reagent method. Furthermore, the RNeasy Mini Kit provided RNA samples with superior purity, as evidenced by a higher 260/230 ratio. Although RNA extracted using the TRI Reagent exhibited slightly elevated mRNA expression levels for the three biomarkers, these differences did not reach statistical significance. In summary, both methods are suitable for total RNA extraction, but the RNeasy Mini Kit is recommended for obtaining higher RNA concentration and purity. The choice of method should be contingent upon the intended downstream application of the RNA.

A cost-effective RNA extraction and RT-qPCR approach to detect California serogroup viruses from pooled mosquito samples

Mosquito-borne diseases pose ongoing global health concerns, demanding more cost-efficient methods to detect pathogens to support enhanced surveillance efforts. This study introduces an adapted TRIzol-based high-throughput RNA extraction protocol, tailored for the detection of California serogroup viruses in pooled mosquito samples in a rapid and cost-effective manner. This approach provided consistent RNA yields and sensitive viral detection relative to two commercial extraction kits (QIAGEN RNeasy Mini Kit and MACHEREY–NAGEL NucleoSpin RNA Plus Kit). The incorporation of a user-friendly and non-spiking-based RT-qPCR internal control designed for the 18S rRNA gene in mosquitoes minimizes potential false positives/negatives, improving the fidelity of viral detection outcomes. Effective RNA yields, purity, and successful target amplification across 25 mosquito species and varied pool sizes (1–50 mosquitoes per tube) affirm the reliability of our approach. The extraction method is cost-effective, with an incurred cost of $0.58 CAD per sample, in contrast to the $5.25 CAD cost per sample of the two kits, rendering it promising for mosquito-borne disease surveillance initiatives.

Association of serum level and gene expression of IL-25 in a sample of Iraqi asthmatic patients

Asthma is an increasingly common disease characterized by allergic airway inflammation, hyperresponsiveness, mucus overproduction, and peribronchial fibrosis. The epithelial cytokines IL25 have been implicated in asthma pathogenesis because they promote Th2-type cytokine synthesis. The current study aimed to assess IL-25 expression and measure the serum level of this cytokine in asthmatic Iraqi patients. The study was conducted on sixty (60) asthmatic patients (32 female and28 male) and thirty as apparently healthy subjects (control) (16 female and 14 male) with ages ranging from 15-60 years recruited Alzahra’a Center for Asthma and Allergy in Baghdad during the period between November /2021 to March /2022. Written informed consent was obtained from all patients and a healthy control group. Moreover, the study design was approved by the Institute of Genetic Engineering and Biotechnology for Postgraduate Studies/ University of Baghdad. All patients were diagnosed according to clinical examination by a chest physician and selected according to the global initiative of asthma criteria. The healthy control group are non-smoking subjects with no history of obstructive lung disease or atopy. The RNA was extracted from the whole blood of asthma patients and healthy control without Trizol subjects using the protocol in Easy Pure t® blood RNA Kit (ER-401). The acceptable purity of asthmatic patients and control healthy group RNA ranges between 1.84 and 1.99. IL-25 gene expression showed the Ct of asthma patient group (25.179) and control group (27.393), and the2-ΔΔCt of asthma patient group (288.81) and control group (58.48) and the fold of gene expression was statistically significantly (P≤0.01) the calculation of the gene expression it was significant in the patient group than control group4.938 times with ratio (4.938:1) . ELISA estimated IL-25concentration, the results were statistically significant (P≤0.001), the level of IL-25 in asthma patient group (29.30± 5.107317pg/ml) while the level of IL-23 in Control group (15.16±0.085248pg/ml). Keywords: IL-25, gene expression, Elisa, Asthma

EVALUATION OF METHODS FOR TOTAL RNA EXTRACTION FROM THE ENDOSPERM OF COCOS NUCIFERA VAR. MAKAPUNO IN VIETNAM FOR MOLECULAR ANALYSIS

Sap coconut (Cocos nucifera L. var. makapuno) in Vietnam is a mutant coconut variant; coconut water is in the state of lotus glue, and coconut rice is like cream. For high-quality transcriptome, sequencing, quality, purity and concentration of RNA are the key factors. However, coconut endosperm tissue has higher stiffness and fatness than the leaf tissue, which complicates the extraction process. Moreover, RNA is much more difficult to preserve than DNA. In this study, various RNA extraction methods were examined in Vietnamese waxy coconut endosperm tissue samples. Optimum extraction and preservation of the RNA using the simplest possible chemicals was the objective of this study. The modified CTAB method with LiCl and the TRI reagent method were tested and evaluated. The purity, concentration and quality of RNA after storage were improved. The findings indicated that the TRIsure extraction method with the addition of NaCl and β-mercaptoethanol yielded optimum RNA quality. The RNA concentration was 159 ng/µL, with a purity ratio of 1.94 ± 0.04 for A260/A280 and 1.58 ± 0.02 for A260/230. RNA samples remained stable for up to 3 weeks when stored in absolute ethanol at 8°C–10°C, which significantly reduced their degradation during transportation. This study facilitated the use of simple chemicals for high-quality RNA extraction from coconut endosperm and its preservation for applications in high throughput sequencing.

PSII-6 Changes in Galectin Gene Expression and Secretion in Response to Gum Arabica Drench in Goats

Abstract Acacia senegal is part of the natural diet of goats. Gum Arabica (GA) is a water-soluble complex polysaccharide antimicrobial, anti-inflammatory prebiotic, derived from Acacia Senegal. Its mechanism of action has not been defined. Galectins are secreted sugar-binding animal proteins involved in the regulation of innate and adaptive immunity. They function in microbe recognition and as regulators of inflammation. This study evaluated the effect of GA on Galectin transcription. Clinically healthy Boer X Spanish goats (n = 20) were randomly assigned to two groups of 10. Goats in the treatment group received 10 mL of GA in water daily for six weeks, controls received sterile water. Blood was collected weekly from the jugular vein using acid citrate dextrose as an anticoagulant. Subsequently, the blood was centrifuged to isolate the cells and plasma. Total RNA isolation was isolated from the cell pellet using Trizol (Sigma-Aldrich). The RNA concentration (ng/μL) and purity was assessed using a Nanodrop Spectrophotometer (Thermo Scientific Inc). Galectin-1, -3 and -9 transcription was conducted using goat specific primers and housekeeping genes for real-time PCR. Fold changes were calculated using the Livak method. The Log2FC values for Galectin 1, 3, and 9 expressions were 12.47, 0.29, and 2.22 respectively. Thus, GA had varying effects on structurally distinct galectins. Galectin 1 gene (Lgals1) is an important modulator of the immune response and was highly induced. Changes in Galectin transcription can impact microbial recognition and immune activation for goat health. Further analyses are needed to evaluate the functional effects of GA for application as a prebiotic and immune modulator lain goat health.

PSII-3 Sheep Neutrophil Activation in Response to Garlic in Vitro

Abstract St. Croix hair sheep are known to resist parasitic infection. Parasitic resistance is in part due to the increased number and ability of neutrophil leukocytes to fight pathogens. Dietary supplements such as garlic (Allium sativum L.) are antimicrobial immune modulators that can enhance production. This study sought to evaluate the impact of fresh filtered garlic on sheep neutrophils through In vitro treatment. The study collected blood from the jugular vein of five clinically healthy female St. Croix sheep at NC A&amp;T State University Farm and isolated neutrophils from 10 mL whole blood samples from each animal. The neutrophils were treated with fresh filtered garlic after the total cell count was measured, and 1 million cells were taken for treatment. The total protein concentration in the neutrophil supernatant was measured before and after treatment. Garlic (10 ug/mL PBS) was added to neutrophils, incubated (30 minutes, 37 °C) and centrifuged. Total RNA was extracted from the pellet using the TRIZOL RNA Isolation Protocol (Sigma-Aldrich). The RNA concentration and purity were assessed using a Nanodrop Spectrophotometer (Thermo Scientific Inc.). Pooled RNA from each group was used for cDNA synthesis, followed by qPCR analysis of the expression of 84 test genes related to the innate and adaptive immune response (RT2 profiler array) of the host. Total protein concentration in the supernatant was determined using the BCA Assay (Pierce BCA Protein Assay Kit). In vitro treatment with garlic extract resulted in increased total secreted protein concentrations from treated neutrophils, as well as upregulation of 10 genes involved in immune response and downregulation of 4 genes associated with inflammation and immune regulation. The study findings indicate that garlic stimulates gene transcription and protein secretion in neutrophils from St. Croix sheep in vitro. Studies are needed on the possible impacts on neutrophil function and animal response to infection for sustainable use of garlic in animal production.