NanoDrop Spectrophotometer Research Articles

Association of HPA Antigens with Immune Thrombocytopenia: A Case-Control Study by PCR-SSP Method

Background: Human platelet antigens (HPAs) play a clinically significant role in alloimmunization and the development of immune-mediated disorders such as immune thrombocytopenia (ITP), fetal and neonatal alloimmune thrombocytopenia (FNAIT), and post-transfusion purpura (PTP). Understanding the genetic profiles of HPAs is critical for preventing and treating these conditions. Given the limitations of serological methods in determining HPA genotypes, this study aims to investigate the association between the genotypes of HPA1, HPA2, HPA3, HPA4, and HPA15 antigens and autoimmune thrombocytopenia in Lorestan Province, utilizing the PCR-SSP method. Materials and Methods: This case-control study involved 80 individuals diagnosed with ITP and 120 healthy controls. DNA samples were extracted using a commercial DNA extraction kit, with concentrations quantified via a Nanodrop spectrophotometer. Genotyping was performed using the PCR-SSP method with specific primers for each HPA gene. The genotype data were verified using previously established sample sets. The frequencies of each HPA genotype were recorded, and a comparative analysis was conducted between the patient and control groups to evaluate the study hypothesis. Results: The results revealed that individuals carrying the HPA2b allele had a 5.31-fold increased risk of developing ITP, a statistically significant finding (P < 0.05, OR = 5.31). Similarly, the presence of the HPA15b allele was associated with a 6.54-fold increased risk (P < 0.05, OR = 6.54). Conclusion: These findings, in conjunction with previous studies, suggest the need for larger-scale investigations across different populations. Such research could aid in the early diagnosis and prediction of thrombocytopenia severity, inform treatment strategies, and facilitate the removal of pathogenic antibodies from circulation.

Targeted microscopic rare cell retrieval yields sufficient deoxyribonucleic acid for molecular testing in fixed tissue with low tumor fraction

Abstract Introduction/Objective DNA extracted from formalin-fixed paraffin-embedded tissue (FFPE) blocks is often utilized for molecular oncologic testing. Cases with low tumor fraction often fail to obtain adequate tumor for molecular testing. We hypothesized that targeted retrieval of visualized tumor cells from FFPE sections, including those with low tumor fraction, could yield sufficient DNA for molecular testing. Methods/Case Report Unstained sections of various thicknesses were cut from low tumor fraction blocks and visualized by light microscopy and the RareCyte CyteFinder II (Seattle, WA). Microscopic cell retrieval was performed on the RareCyte using 40 µm diameter needles from identified regions of interest. Samples were deparaffinized using Zymo Research (Tustin, CA) solution, and digested and purified using Zymo-Spin Column technology. Sample DNA was quantified with NanoDrop Spectrophotometer (Chino, CA). Results (if a Case Study enter NA) Regions of interest were morphologically identifiable on 4-10 µm thick sections and harder to visualize on 12 µm thick sections. Targeted microscopic cell retrieval was performed with 40 µm diameter needles. Retrieval was performed from 26 to 34 regions of interest per sample and pooled for molecular testing. Samples (n=4) yielded a mean of 81.0 ng of DNA ± 18.4 ng (1.8 ng/µL ± 0.4 ng/µL) and a median of 87.75 ng (1.95 ng/µL). Conclusion Targeted microscopic cell retrieval from low tumor fraction FFPE slides using the RareCyte CyteFinder II can provide sufficient DNA material for molecular testing. Application of this technology has the possibility of recovering sufficient tumor DNA for molecular testing from specimens that may be deemed unsuitable for molecular testing according to current standards. We aim to focus on increasing sample size and completing comprehensive molecular analysis as we continue to expand this work.

The Rule of CTLA-4 +49 G/A Gene Polymorphism Association With T2DM

The existing study has been designed to consider relationship among polymorphism of CTLA-4 +49 A/G (rs231775) in patients through diabetes mellitus type 2 (T2D). The training has stayed directed on adult at the subdivision of College of Medicine, AlQadisiya University throughout the period prolonged as of October, 2023 to April, 2024. Fifty patients with 50 control healthy assigned to 2 equal groups, collection blood samples with EDTA tubes. The study enrolled 50 Type 2 Diabetes patients and 50 healthy subjects, with no important variances in age, gender, or residency. Demographic appearances displayed no important alteration among the two groups. Chronic illnesses, such as diabetes mellitus and systemic hypertension, were found to be highly significant between patients and control subjects. Hypertension rates were higher in patients than in control subjects, while liver disease rates were higher in patients. Heart attacks were upper in sick than in controlling issues, and hyperlipidemia rates were higher in patients. Drug types and treatment periods were not statistically significant among T2D patients and healthy control. Genomic DNA stayed removed from blood testers by means of a gSYAN DNA kit withdrawal kit and checked using a Nano drop spectrophotometer. The polymorphism of CTLA-4 +49 A/G (rs231775) displayed that the AG genotype and the A allele were more frequent in the type 2 diabetes patients compared to controls, indicating they may be risk factors for developing T2D.

Association of the vitamin D metabolism gene polymorphism with the severity of coronary lesions assessed by SYNTAX score

This study aimed to determine the association of vitamin D serum blood levels and vitamin D gene polymorphism with the severity of coronary lesions in patients with stable coronary artery disease (CAD). Material and methods. 260 patients with stable CAD (average age was 58 years) were examined in the presented research. All patients were divided into two groups according to the SYNTAX score: low-risk patients with SYNTAX score ≤ 31 (n = 224) and high-risk patients with SYNTAX score > 31 (n = 36). For enzyme-linked immunosorbent assay and genetic analysis, peripheral blood was collected from the cubital vein into vacuum tubes containing coagulation activator and K3-EDTA, respectively. Serum blood level of 25-hydroxyvitamin D (DiaSource Diagnostics, Belgium) and 1,25-dihydroxyvitamin D (Immunodiagnostic Systems, Great Britain) were determined by enzyme-linked immunosorbent assay according to the manufacturers’ protocols. Genomic DNA was isolated by phenol-chloroform extraction method from whole blood. The quality and quantity of isolated DNA were assessed using NanoDrop spectrophotometer (Thermo Fisher Scientific, USA). Five polymorphic variants in the VDR (rs2228570 and rs73123) and GC (rs7041, rs1155563 and rs2298849) genes were selected for analysis. Genotyping was performed by real-time PCR in a 96-well plate with fluorescently labeled TaqMan probes. The quality of PCR was controlled by repeated genotyping of 10 % of the analyzed samples. Results. We found no statistically significant differences in serum blood level of the studied markers in patients from low-risk and high-risk groups. One polymorphic variant in the GC gene associated with the multiple coronary lesions (rs2298849) (odds ratio 2.26, 95 % confidence interval 1.28–3.99, p = 0.006) according to an additive inheritance model was identified. In addition, we determined the association between low serum blood level of 1,25-dihydroxyvitamin D in patients with CAD with multiple lesions of the coronary vascular system with A/A – A/G genotypes of the rs2228570 polymorphism in the VDR gene, A/A genotype of the rs7041 polymorphism and A/A genotype of the rs2298849 polymorphism in the GC gene. Conclusions. Allelic variants in the vitamin D metabolism genes are associated with the degree of coronary artery lesions assessed by the SYNTAX score in patients with stable CAD. Also, serum blood level of the active form of vitamin D (1,25-dihydroxyvitamin D) is less in carriers of homozygous genotypes for the major alleles of the VDR and GC genes.

Simultaneous Extraction of Bone Marrow RNA and DNA from Patients with Hematologic Diseases Using a Combined Magnetic Bead Method within 1 Hour.

TRIzolTM is widely used for RNA and DNA extraction. However, this method is laborious and time-consuming. The objective of this study was to validate a time-effective and labor-saving protocol. The TRIzol method was used to separate the aqueous phase, protein, and phenol layer of bone marrow samples from 12 patients with hematological diseases. Subsequently, RNA and DNA were extracted from the aqueous layer containing RNA and phenol layer containing DNA, respectively, using magnetic bead extraction kits. The quantity and purity of extracted RNA and DNA were examined using a NanoDrop spectrophotometer. Quantitative fluorescence PCR amplification of the ABL1 gene was performed to verify the effectiveness of the extracted RNA and DNA for downstream experiments. RNA and DNA from another 16 bone marrow samples were extracted to compare the performance of the two methods. Co-extraction of RNA and DNA was completed within 1 h. The data showed that RNA and DNA yield ranged from 13.1 to 204.5 ng/µL and 33.1 to 228.8 ng/µL, respectively. The A260/A280 ratios of RNA and DNA samples ranged from 1.82 to 2.01 and 1.73 to 1.91, respectively. RNA and DNA extracted using this scheme exhibited ideal performance in quantitative fluorescence PCR. The present protocol showed better quality and effectiveness in extracting RNA and DNA compared to the TRIzol method. This protocol for RNA and DNA co-extraction is fast, labor-saving, and high throughput. It can be adopted for routine molecular biology analyses, particularly for non-reproducible specimens.

A Comparison Study of Cryopreservation of Seminal Fluid between Flinders Technology Associates (FTA-card) and Swab for Multiplex Short Tandem Repeats (STR) Analysis

Background Whatman™ FTA (Flinders Technology Associates) is a form of card soaked in chemicals that denatures proteins while protecting DNA and ensuring the safe handling of dried body fluid spots (blood, semen, and saliva) and buccal cells. To this day, these cards are still infrequently used in forensic science. Therefore, reference samples including biological material may be collected on FTA cards for genetic analysis that have been widely used for DNA preservation and analysis, particularly in forensic sciences and genetic studies. The goal of the study was to evaluate the effects of cryopreservation on DNA quantitative and short tandem repeats (STR) profiling by comparing the yield and quality of DNA extracted before and after cryopreservation using FTA card and swabs in the forensic analysis. The mean total DNA concentration and the purity of seminal fluid from FTA-cards and swabs before and after sperm cryopreservation were measured by using nano-drop spectrophotometer (93.688 ± 12.5) (90.94 ± 13.9) ng/ µl; ( 221.07 ± 20.97)( 135.47 ± 19.6) ng/ µl respectively. Also a highly significant difference (p<0.001) was detected when the results of concentration DNA on FTA card were compared with dilution using wash media before and after sperm cryopreservation. Whereas significant difference (p<0.05) was observed between DNA concentration on FTA card with and without dilution after sperm cryopreservation. STR loci were present in the majority of data, particularly in FTA-card and swab samples where results explained that 100% of STR profiles were present in all 15 loci, both before and after sperm cryopreservation. In contrast to the conventional method of storing and analyzing STR profiling, it can be concluded that seminal fluid samples taken by FTA card and swabs can be taken and stored for a time by cryopreservation, leads to maintaining DNA integration in the field of criminal research.

Study of the DNA Extraction from the Nail by Spin Column-based Nucleic Acid Purification

Abstract Background: Nails are one of the objects that are more durable than other witness objects. This study was interested in whether the Spin column-based nucleic acid purification technique could extract DNA from nails to give satisfactory results. Aims and Objectives: This study aimed to evaluate the yield and quality of DNA extracted from the nail. Materials and Methods: Nail samples from 15 males and 15 females over 18 were extracted using GeneAll® Exgene™ Cell SV Mini Kit. The DNA concentration and purity were measured using a NanoDrop Spectrophotometer, and the quality of DNA was assessed by polymerase chain reaction (PCR) using primers specific for human growth hormone (HGH). Results: The average yield of DNA was 0.816 μg (0.141–2.706 μg) obtained from the average nail weight of 30.2 mg (22.2–40.0 mg). The average DNA concentration was 27.2 ng/μL (4.7–90.2 ng/μL), corresponding to 30 μL in volume. Almost all DNA samples (96.7%) had high purity (A260/A280 ≥1.80) and gave a band of PCR product of the HGH gene on agarose gel electrophoresis. Conclusion: Spin column-based nucleic acid purification is recommended for nail DNA extraction due to its simplicity and high quality.

Identification of MiR398 and Its Regulatory Roles in Terpenoid Biosynthesis of Persicaria odorata

Persicaria odorata is an herbaceous plant with antifungal and antibacterial properties. The plant produces secondary metabolites, including phenols, sulphur-containing compounds and terpenoids, in response to biotic and abiotic stresses. Terpenoids in P. odorata are synthesized through the mevalonate (MVA) and the methylerythritol phosphate (MEP) pathways. 1-deoxy-D-xylulose-5phosphate isomerase (DXR) is a rate-limiting enzyme in the MEP pathway and may be regulated by microRNA (miRNA) miR398. There is a lack of evidence showing miR398 regulation in terpenoid biosynthesis in P. odorata through DXR-targeting. The study aimed to verify the stem-loop structure of miR398 and analyse its expression towards the target gene, DXR, in treated and control P. odorata. RNA was quantified and qualitatively analysed using a Nanodrop spectrophotometer and agarose gel electrophoresis. The stem loop of miR398 was verified using reverse transcriptase PCR (RT-PCR) and agarose gel electrophoresis. The expression of miR398 and DXR was compared between control and treated samples. Treated leaves were punctured with needles and left for 48h before harvest. Gene expressions were quantified and normalised using reference genes. The stem-loop structure of miR398 was confirmed, despite possible primer mismatches and unspecific binding. This step was essential before comparing and assessing the gene expression of miR398 and DXR. A decrease in abundance of miR398 whereas in increase in abundance was in the treated sample compared to the control sample indicating that miR398 negatively regulated DXR under stress conditions, suggesting an increase in terpenoid synthesis as a defence mechanism. DXR acts as a rate-limiting enzyme in the MEP metabolic pathway. Further studies are needed to quantify the effects of miR398 on terpenoid biosynthesis after wounding in P. odorata.

A Comparison of Different Protocols for the Extraction of Microbial DNA Inhabiting Synthetic Mars Simulant Soil.

Compared with typical Earth soil, Martian soil and Mars simulant soils have distinct properties, including pH > 8.0 and high contents of silicates, iron-rich minerals, sulfates, and metal oxides. This unique soil matrix poses a major challenge for extracting microbial DNA. In particular, mineral adsorption and the generation of destructive hydroxyl radicals through cationic redox cycling may interfere with DNA extraction. This study evaluated different protocols for extracting microbial DNA from Mars Global Simulant (MGS-1), a Mars simulant soil. Two commercial kits were tested: the FastDNA SPIN Kit for soil ("MP kit") and the DNeasy PowerSoil Pro Kit ("PowerSoil kit"). MGS-1 was incubated with living soil for five weeks, and DNA was extracted from aliquots using the kits. After extraction, the DNA was quantified with a NanoDrop spectrophotometer and used as the template for 16S rRNA gene amplicon sequencing and qPCR. The MP kit was the most efficient, yielding approximately four times more DNA than the PowerSoil kit. DNA extracted using the MP kit with 0.5 g soil resulted in 28,642-37,805 16S rRNA gene sequence reads and 30,380-42,070 16S rRNA gene copies, whereas the 16S rRNA gene could not be amplified from DNA extracted using the PowerSoil kit. We suggest that the FastDNA SPIN Kit is the best option for studying microbial communities in Mars simulant soils.

Concentration of isolated DNA face masks made of gelatin for halal authentication

The function of using a face mask is to remove dirt and dead skin cells found in the pores of the skin, open clogged pores and clean the remaining cosmetics that cannot remove with ordinary cleansers, repair and tighten the skin, provide nutrition, smooth, soften and maintain skin moisture. People are aware of halal-labeled cosmetics, one of which is face masks because some face masks contain gelatin. Some face masks on the market contain gelatin. Preparation of 5 reference samples with different concentrations. Preparation of face masks, DNA isolation, and checking the isolation results. The test results with the Nanodrop Spectrophotometer showed that the face mask DNA containing pig and cow gelatin extracted in this study ranged from 163.35 ng/µl in sample S6 to 83.550 ng/µl in sample S7. With the largest concentrations of 163.35 ng/µl and 83.550 ng/µl, while the highest purity value at the λ260/λ280 ratio was 1.41.

DNA quality and quantity in adipose tissue: a comparison of the effects of bomb explosion

BACKGROUND Adipose tissue is often overlooked in DNA testing due to misconceptions about its DNA content. However, its shock-absorbing qualities may be useful for high-pressure scenarios like bomb blasts. This study aimed to evaluate DNA quality and quantity in adipose tissue affected by blasts compared to that in unaffected tissue.
 METHODS 10 adipose tissue samples were taken from regions near and far from the blast, representing the blast-exposed and non-blast-exposed groups. The adipose tissue was stored at a low temperature for 5 days, after which an organic extraction method was applied. The purity of the DNA extract was assessed using a NanoDrop spectrophotometer, and its integrity was evaluated using 0.8% concentration gel electrophoresis at 60 V for 90 min. DNA typing was conducted using the GlobalFiler™ kit, and DNA quantity was determined with the Quantifiler™ Trio DNA Quantification kit.
 RESULTS Of 20 DNA extracts from adipose tissue, all samples demonstrated purity, integrity, and complete typing results. Adequate integrity was found in 90% of samples in both groups. A 50% incidence of allele shifting was observed at the D7S820 locus within the blast-exposed group.
 CONCLUSIONS DNA from blast-exposed adipose tissue exhibited no significant quality or quantity differences from non-blast-exposed tissue. This suggested adipose tissue’s potential as an alternative DNA source in a bomb explosion.

Salivary Protein Quantification among Periodontitis Patients with Different Disease Severity: A Pilot Study

The salivary protein quantification reflecting the periodontal pathophysiology among patients with different periodontitis severity has not been well explored. This quantification is fundamental as the changes in the quantity of proteins in different severities of periodontitis can reflect the periodontal complex alterations. This pilot study aimed to quantify and compare the salivary proteins by collecting unstimulated saliva from periodontitis patients and healthy individuals. The radioimmunoprecipitation assay (RIPA) protocol was used to extract protein from patients’ saliva and quantified using the Nanodrop Spectrophotometer 2000/2000c (A280) set at 280 nm wavelength. The mean concentration of salivary proteins was shown to be 26.48 + 2.95 mg/mL in healthy patients, 22.00 + 0.38 mg/mL in Stage 1 periodontitis patients, 27.34 + 1.61 mg/mL in Stage 2 periodontitis patients, 27.11 + 0.66 mg/mL in Stage 3 periodontitis patients and 24.89 + 1.91 mg/mL in Stage 4 periodontitis patients. However, the mean difference between all groups was not statistically significant. Within the limitations of this study, there was no difference in the salivary protein quantity among patients with different stages of periodontitis.

Protocol Development for Yielding High Quality DNA and RNA from Archived Formalin-Fixed Paraffin Embedded Tissues

Introduction: While genetic analysis of archived formalin-fixed paraffin embedded (FFPE) tissue specimens would be a significant research resource, extraction of high-quality DNA and RNA from these specimens is a significant challenge. Storage duration, tissue handling and tissue preservation processes as well as their interactions with the nucleic acids could influence the quality and integrity of the DNA or RNA required for genetic analysis.
 Objectives: The goal of this study was to establish a protocol to extract high quality DNA and RNA from the FFPE tissues for further use in quantitative PCR analysis.
 Methods: For the purposes of this study, human FFPE biopsy tissues from lung, liver, colon and kidney were obtained from a single center biorepository. GeneJET Genomic DNA Purification Kit (Catalog # K0722) obtained from Thermo Fisher Scientific and RecoverAll Total Nucleic Acid Isolation Kit obtained from Life Technologies were modified and used for extraction of DNA and RNA, respectively. Briefly, modifications involved extending reagent incubation times, increasing sample volumes and wash steps, and increased final nucleic acids recovery and concentration steps. Eight sections around 8-10 ?m thick were microtomed for each tissue sample and used for extraction. The purity of the nucleic acids obtained was verified using Nanodrop Spectrophotometer.
 Results: The average DNA yield from eight sections for each of the tissues was 270±184 ng/?l and for RNA was 296±188 ng/?l. Nucleic acid quality was assessed by measuring the 260nm/280nm absorbance ratio for protein contamination as well as the 260nm/230nm absorbance ratio for salt contamination. Both were found to be within acceptable ranges. RNA was reverse transcribed to cDNA and qPCR was successfully performed on both DNA and cDNA samples.
 Conclusions: These results indicate that protocols using the silica-based membrane technology can yield high quality DNA and RNA that can be successfully used for downstream genetic analysis.

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.

PSV-26 Use of Garlic Drench in the Modulation of Galectin Gene Expression in St Croix Sheep

Abstract St. Croix sheep are parasite resistant and can live in a wide variety of climates. Dietary supplements can influence innate immunity. Garlic (Allium sativum) is used as an immune modulator and an alternative antibiotic. Lectins in garlic could interfere with animal lectins such as Galectins (Gals). Galectins bind β-galactosides and modulate pathological processes and immunity. The effect of garlic drench on Gal (1,3,9) gene expression in sheep blood was evaluated. All protocols were approved by the IACUC. Clinically healthy, adult St. Croix sheep were divided into two groups. The treatment group (n = 6) was drenched with 5 mL garlic barrier at a rate of 10 mL diluted in sterile distilled water 1:1. The control group (n = 6) was drenched with 10 mL sterile distilled water for four weeks. Cells and plasma were separated by centrifugation of whole blood. The concentration of total plasma protein was determined using the BCA assay kit (Pierce). Secretion of Galectins 1,3,9 was evaluated using Sheep-specific ELISA(My-BioSource). Data were analyzed using SAS Statistical analysis software 9.4 (SAS Institute). Nucleic acid purity and concentrations were assessed using the Nanodrop spectrophotometer. Galectin gene transcription was evaluated using sheep Gal 1,3 and 9 specific primers commercially sequenced (Eurofins Genomics). The house keeping genes, Ribosomal Protein Lateral Stalk Subunit P0 and Ubiquitin C-Terminal Hydrolase L5, were used as internal controls. Samples were amplified in duplicate using the CFX Connect real-time PCR system. The Livak Method was used to calculate the fold change in gene expression. Galectin 1, 3, and 9 were transcribed and translated. Transcription and translation were differentially affected by garlic. Galectin 1 transcription increased; secretion decreased. Galectin 3 transcription and secretion increased at P < 0.05. There was no change in the transcription of galectin 9, however, secretion was increased. Thus, garlic drench deferentially impacted transcription and secretion of structurally distinct Gal in sheep blood. Further studies are needed to determine the effect of Garlic on Gal-mediated functions important to sheep health and production.

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&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.

Efficiency of DNA Extractions Methods from Pigeons and Cockatiels Feathers

This study aimed to compare different DNA extraction methods to achieve higher amounts and purity levels from molted feathers of pigeons (Columba livia f. domestica) and cockatiels (Nymphicus hollandicus). We evaluated 226 animals consisting of 202 pigeons and 24 cockatiels for these purposes. We performed three commercially available DNA extraction kits to isolate DNA from the feather samples. These kits were compared regarding DNA yield and quality depending on the different applications made during the isolation. DNA concentration (ng/mL) and absorbance ratio (260/280) were measured using a Nanodrop spectrophotometer. Kruskal-Wallis test with the Dunn’s post hoc comparison was performed for the statistical comparisons. The mean DNA concentration was the highest in isolation with the kit C. Among three commercial kits, statistically significant differences were observed concerning nucleic acid concentration (ng/µL) (P

Evolution of TLR3 gene in Nigerian indigenous and exotic turkeys

This experiment was carried out to determine the evolution of the TLR3 gene in Nigerian indigenous and exotic turkeys. A total of 150 turkeys i.e. 100 exotic and 50 indigenous were used for this study. Birds were allotted treatments and fed formulated grower diets that meet the NRC—Turkey grower nutritional requirements. Genomic DNA was isolated from each blood sample, and the concentration and purity of each DNA sample was determined using a Nanodrop Spectrophotometer. After DNA extraction and quantification, three DNA samples from each genetic group (or breed) were taken for TLR3 gene discovery and polymerase chain reaction (PCR). The source sequence and the retrieved sequences were trimmed and edited while the amino acid sequences of the new turkey TLRs were predicted based on the open reading frames of the expressed nucleotide sequences, and the nomenclatures of turkey TLRs were based on the best hits of the proteins in the database. From the findings of the study, the SNP (172N>N) detected at position 516 was synonymous in both exotic and local turkey breeds, and this resulted in amino acid changes from Alanine to Threonine in exotic turkeys and Cysteine to Tyrosine in local turkeys. Also, in this study, the SNP (7 Q>*) detected in exon 4 was non-synonymous, resulting in amino acid changes from Cytosine to Threonine in the exotic turkey. Positive Tajima's D values were the same for exotic and local turkey breeds. The findings of this study suggest significant instances of purifying selection acting on the gene. Based on the results of this research, the populations of local and exotic turkeys showed a low level of heterozygosity at TLR3 locus and may be homogenous. Also, there was low genetic diversity in the TLR3 gene of both local and exotic turkey breeds. This suggests potential vulnerabilities and challenges for their long-term health and adaptability. Strategies that aim to increase genetic diversity, like cross-breeding should be implemented.

Analysis of Purity and Concentration Escherichia coli DNA by Boiling Method Isolation with Addition of Proteinase-K and RNase

Escherichia coli is a leading cause of Urinary Tract Infections (UTIs) in Indonesia, with approximately 180,000 cases reported annually. The more cases of UTIs, the more PCR diagnosis is needed with an accurate, fast, simple, and economical DNA isolation method. However, currently, there is no DNA purification stage from protein and RNA contaminants in the boiling DNA isolation method. This study aimed to investigate the impact of incorporating Proteinase-K and RNase into the boiling DNA isolation method on the purity and concentration of E. coli’s DNA during isolation. The boiling method involved heating to 95°C – 100°C bring to cell lysis and release of cellular components, including DNA. Urine samples were artificially contaminated with E. coli at different McFarland standards (0.25, 0.5, and 1). The boiling DNA isolation method was then performed and then analyzed for purity and concentration using a NanoDrop spectrophotometer. This study demonstrated a positive correlation between Proteinase-K and RNase concentrations used in the boiling DNA isolation method and the subsequent increase in DNA purity and concentration. An increase in DNA purity and concentration was obtained even though it was not statistically significant compared to that without Proteinase-K and RNase addition, with p-values of 0.245 for DNA purity and 0.353 for DNA concentration. Further research is recommended with higher Proteinase-K and RNase concentrations in the boiling DNA isolation method to achieve improved purity and concentration of E. coli DNA. Such enhancements could improve PCR amplification and help diagnose E. coli-related UTIs.

Comparison of Purity and Concentration Values of Mycobacterium tuberculosis DNA Extraction Result from the Boiling and Spin Column Method

The Polymerase Chain Reaction (PCR) technique is extensively employed in molecular biology to precisely detect Mycobacterium tuberculosis. Prior to conducting PCR, extracting of high-quality genomic Deoxyribonucleic Acid (DNA) is crucial to ensure accurate and reliable results. The primary objective of this study is to conduct a comparative analysis of the purity and concentration of M. tuberculosis DNA acquired through the utilization of the boiling method and the spin column extraction methods. A descriptive comparative research design was employed, utilizing a sample of 16 sputum specimens that had previously been confirmed as positive for M. tuberculosis through Acid-Fast Bacteria (AFB) examination and Molecular Rapid Test (MRT). The extraction of DNA was carried out using the boiling method and the spin column method. Subsequently, the concentration and purity of the extracted DNA were assessed using the NanoDrop Spectrophotometer, and the results were compared. The obtained yield of M. tuberculosis DNA isolates through the boiling method ranged from 9.6 ng/µL to 1258.7 ng/µL, with an average purity value of 1.23. Conversely, for the spin column method, the concentration of M. tuberculosis DNA isolates ranged from 8.7 ng/µL to 207.8 ng/µL, with an average purity value of 1.83. In conclusion, there is a significant difference between the purity and concentration of M. tuberculosis DNA extraction results using the boiling method and spin column methods.