NanoDrop Spectrophotometer Research Articles

Quality and quantity of dromedary camel DNA sampled from whole-blood, saliva, and tail-hair.

Camels are livestock with unique adaptations to hot-arid regions. To effectively study camel traits, a biobank of camel DNA specimens with associated biological information is needed. We examined whole-blood, saliva (buccal swabs), and tail-hair follicle samples to determine which is the best source for establishing a DNA biobank. We inspected five amounts of each of whole-blood, buccal swabs, and tail-hair follicles in nine camels, both qualitatively via gel electrophoresis and quantitatively using a NanoDrop spectrophotometer. We also tested the effects of long term-storage on the quality and quantity of DNA, and measured the rate of degradation, by analyzing three buccal swab samples and 30 tail-hair follicles over a period of nine months. Good quality DNA, in the form of visible large size DNA bands, was extracted from all three sources, for all five amounts. The five volumes of whole-blood samples (20–100μl) provided ~0.4–3.6 μg, the five quantities of buccal swabs (1–5) produced ~0.1–12 μg, while the five amounts of tail-hair follicles (10–50) resulted in ~0.7–25 μg. No differences in the rate of degradation of buccal swab and tail-hair follicle DNA were detected, but there was clearly greater deterioration in the quality of DNA extracted from buccal swabs when compared to tail-hair follicles. We recommend using tail-hair samples for camel DNA biobanking, because it resulted in both an adequate quality and quantity of DNA, along with its ease of collection, transportation, and storage. Compared to its success in studies of other domesticated animals, we anticipate that using ~50 tail-hair follicles will provide sufficient DNA for sequencing or SNP genotyping.

Studying Methylation Status of Protein Tyrosine Phosphatase Receptor Gamma (PTPRG) in Chronic Myeloid Leukemia Patients in the State of Qatar

Background:Chronic myeloid leukemia (CML) is a clonal myeloid stem cell disorder associated with increased myeloid cells spectrum in peripheral blood. The disease is due to the genetic translocation t(9;22)(q34;q11.2) encoding for the BCR-ABL1 fusion oncogene which is the hallmark of CML. The resultant chimeric protein P210 kD of BCR-ABL1 causes a constitutive activation of tyrosine kinase domain that leads to continuous activation of several cell signaling pathways.The CML is currently treated with several generations of tyrosine kinase inhibitors (TKIs). However, treatment failure, resistance and disease progression are still reported and the molecular mechanisms still remain poorly understood.On the other hand, protein tyrosine phosphatase receptor gamma (PTPRG) is the natural regulatory mechanism to tyrosine kinase family, acts as tumor suppressor gene, well documented to be downregulated in CML patients while its re-expression led to reduced tyrosine phosphorylation and induced apoptosis in CML cells.PTPRG is also an important protein in the JAK/STAT pathway, has a vital role in the downstream of BCR-ABL protein kinase. Several studies suggested that the aberrant DNA methylation is involved in TKIs resistance in CML leading to leukemic clone escape and disease propagation.In different types of tumors, several mechanisms have been suggested for the loss of function of PTPRG activities such as mutations, deletions, or loss of heterozygosity LOH and gene silencing by methylation.Aim:In this study, we aimed to evaluate and characterize the methylation status of PTPRG gene function in 15 CML Patients 1- Group A: CML patients at diagnosis and 2- Group B) CML patients failed treatments according to European Leukemia Net (ELN) 2013 criteria and 10 healthy individuals. (Labeled as Normal in the figure 1)Materials and Methods:Thirty subsequent peripheral blood samples have been collected from the 15 CML patients at diagnosis and at time of failure. Genomic DNA was isolated from 400 ml of blood on EDTA tubes using Maxwell DNA Purification Kits, the quality and quantity of DNA were measured on Nanodrop Spectrophotometer and gel electrophoresis. The methylation-specific PCR and bisulfite-sequencing were adapted, EZ DNA Methylation Kit was utilized for bisulfite-conversion. Methylation Primers were designed to target the promotor and intronic CpG islands of the PTPRG gene. Gradient PCR was performed to detect the two bands of 321bp and 218bp for CpG islands of promoter and intron of PTPRG respectively. Bisulfite-specific PCR technique was carried out to amplify a fragment of bisulfite-converted DNA that has been pre-defined by the designed bisulfite-specific primers.The Methylation PCR amplified product was sequenced via Sanger Sequencing (Applied Biosystems 3130 Genetic Analyzer). The methylation analysis was performed to study the methylation status of the CpG islands using the ESME (Epigenetic Sequencing Methylation) Analysis Software.Results:Results of the methylation in 25 CpG sites of Promoter region of PTPRG:There are significant differences in the mean of methylation status amongst CML patients at diagnosis and healthy individuals of CpG promoter of PTPRG (p value is ˂0.04). However, there are no significant differences in the mean of methylation status between patients failed treatments and other groups (Figure1)Results of the methylation in 26 CpG sites of Intronic region of PTPRG:There are also significant differences in the mean of methylation status amongst all groups (p value is ˂0.003). (Figure1)Discussion and Conclusions:This is the first prospective study to evaluate epigenetic changes of PTPRG gene from CML patients in Qatar. We previously documented that the expression level of PTPRG is inversely correlated with the expression of BCR-ABL1 in CML patients.In this study, the mechanism behind the downregulation of PTPRG in our cohorts of CML patients was explored and the aberrant methylation of PTPRG could be a mechanism associated with CML disease progression and resistance to TKIs.However, further studies are still needed including larger patient's cohort and CML patients responding to the TKIs.Finally, the methylation of PTPRG in the CML patients at diagnosis and failed treatment may allow the identification of new therapeutic targets and using methylation inhibitors could be also suggested to prevent or reverse TKIs resistance and restore TKIs sensitivity. DisclosuresNo relevant conflicts of interest to declare.

Detection of Staphylococcal Species Diversity within Human Skin Microbiome Using a Simple PCR-SSCP Technique


 Objective. The aim of this study was to detect the diversity of Staphylococcus species of a healthy human skin using a simple techniquevPCR-SSCP.
 Methods: Blood samples, saliva, and skin swaps samples were collected from 50 persons from Hilla City - Iraq. The genomic DNA was extracted from these samples using the Bacteria Genomic DNA Kit. The concentrations and purity of DNA extract estimated by NanoDrop spectrophotometer. Polymerase chain reaction – single-strand conformational polymorphism (PCR–SSCP) technique was performed to detect the diversity between Staphylococcal species in the human skin microbiome using a specific primer of the 16SrRNA gene.
 Results: The PCR results, indicated that the Staphylococcal species were found within the ski community, but it's not infected blood and mouth of test healthy individuals. SSCP-heteroduplex patterns of PCR products appeared the presence Staphylococcal species diversity within skin microbiome of test healthy individuals.
 Conclusion: In spite of the PCR-SSCP, heteroduplex method was simple and cheap and appeared the diversity betweenvStaphylococcalspeciesvin the human skin microbiome, but it's not diagnosed the bacterial strains. So these results required to confirm by DNAvsequencingvtechnique.

Abstract 206: The Consensus Molecular Classification (CMS) of primary colorectal tumors and their matched liver metastasis: Investigating the concordance

Abstract Background The Consensus Molecular Subtypes (CMS) identified four different molecular groups in colorectal cancer (1). Coding mutations are concordant between primary tumors (PT) and matched metastasis (MM) while gene copy numbers seem highly discordant (2). This could somehow influence the molecular classification of PT and MM. We therefore aimed to investigate the concordance of the CMS between PT and MM. Material and methods We retrospectively collected 25 fresh matched samples of primary (P) and synchronous liver metastasis (LM) (discovery cohort) and 50 formalin-fixed-paraffin embedded (FFPE) samples of P and synchronous LM (validation cohort) of chemonaive metastatic colorectal cancer patients. All samples contained at least 30% of tumor cells. RNA was extracted from four sections of 5-μm by using the RNeasy FFPE kit (Qiagen). RNA yield was quantified using a NanoDrop spectrophotometer. Extracted RNA was amplified using a TransPLEX C-WTA whole-transcriptome amplification kit. Amplified cDNA was labeled using the Genomic DNA Enzymatic Labeling Kit and hybridized onto Agendia's full genome arrays both according to the manufacturer's instructions. For FFPE samples, no reference channel was used. Gene expression intensities were normalized using Lowess normalization method implemented in Matlab software version R2012a, and the CMS classification was applied as in Guinney et al (1). Results and conclusion Clinical data were currently available for 66% of patients. Median age at the diagnosis was 64 (35-76), 30% of patients were female and 70% were male. 42% of PT were right side located and 58% were left side located. 42% of tumors were well differentiated, 40% moderately differentiated, 15% poorly differentiated and 3% were not classified. 3% of tumors were MSI, 45% were MSS. No data on the MMR status was available (NA) for 52% of the tumors. Mutations in KRAS were detected in 18% of the tumors while 30% were wild type (WT) and 52% NA. BRAF(V600E) mutation was detected in 3% of tumors, 39% were WT and 58 NA. 73% of patients received surgery of both P and LM simultaneously and before starting a systemic treatment. 72% of patients received a first line treatment. The results of the molecular classification will be presented at the conference.

Changes to Extender, Cryoprotective Medium, and In Vitro Fertilization Improve Zebrafish Sperm Cryopreservation.

Sperm cryopreservation is a highly efficient method for preserving genetic resources. It extends the reproductive period of males and significantly reduces costs normally associated with maintenance of live animal colonies. However, previous zebrafish (Danio rerio) cryopreservation methods have produced variable outcomes and low post-thaw fertilization rates. To improve post-thaw fertilization rates after cryopreservation, we developed a new extender and cryoprotective medium (CPM), introduced quality assessment (QA), determined the optimal cooling rate, and improved the post-thaw in vitro fertilization process. We found that the hypertonic extender E400 preserved motility of sperm held on ice for at least 6 h. We implemented QA by measuring sperm cell densities with a NanoDrop spectrophotometer and sperm motility with computer-assisted sperm analysis (CASA). We developed a CPM, RMMB, which contains raffinose, skim milk, methanol, and bicine buffer. Post-thaw motility indicated that the optimal cooling rate in two types of cryogenic vials was between 10 and 15°C/min. Test thaws from this method produced average motility of 20% ± 13% and an average post-thaw fertilization rate of 68% ± 16%.

Large Instrument- and Detergent-Free Assay for Ultrasensitive Nucleic Acids Isolation via Binary Nanomaterial.

Nucleic acid-based diagnostics are widely used for clinical applications due to their powerful recognition of biomolecule properties. Isolation and purification of nucleic acids such as DNA and RNA in the diagnostic system have been severely hampered in point-of-care testing because of low recovery yields, degradation of nucleic acids due to the use of chaotropic detergent and high temperature, and the requirement of large instruments such as centrifuges and thermal controllers. Here, we report a novel large instrument- and detergent-free assay via binary nanomaterial for ultrasensitive nucleic acid isolation and detection from cells (eukaryotic and prokaryotic). This binary nanomaterial couples a zinc oxide nanomultigonal shuttle (ZnO NMS) for cell membrane rupture without detergent and temperature control and diatomaceous earth with dimethyl suberimidate complex (DDS) for the capture and isolation of nucleic acids (NA) from cells. The ZnO NMS was synthesized to a size of 500 nm to permit efficient cell lysis at room temperature within 2 min using the biological, chemical, and physical properties of the nanomaterial. By combining the ZnO NMS with the DDS and proteinase K, the nucleic acid extraction could be completed in 15 min with high quantity and quality. For bacterial cells, DNA isolation with the binary nanomaterial yielded 100 times more DNA, than a commercial spin column based reference kit, as determined by the NanoDrop spectrophotometer. We believe that this binary nanomaterial will be a useful tool for rapid and sensitive nucleic acid isolation and detection without large instruments and detergent in the field of molecular diagnostics.

Developing quality assurance program for total DNA extraction

The extraction of high quality genomic DNA is pivotal for all downstream genetic analyses. To assist laboratories in the quality assurance of their DNA isolation procedure, the RCPAQAP Molecular Genetics discipline developed a DNA extraction proficiency testing program in 2016 and invited six laboratories across Australia to participate in the first trial. A total of 40 DNA extracts from various tissue types were submitted to the RCPAQAP for external quality assessment. Each DNA extract was analysed on a NanoDrop Spectrophotometer (Thermo Fisher Scientific) and a Quantus Fluorometer (Promega) for concentration and purity determination, and on a DNA TapeStation (Agilent Technologies) for total DNA integrity characterisation. Evaluation of each DNA extract was based on the DNA Integrity Number (DIN), generated by DNA TapeStation, and was used for population Z-score analysis. DNA extracts with a Z-score within two standard deviations of the mean data value were considered good quality. An overview of the laboratory performance in genomic DNA extraction and the associated methods will be presented.

Comparison of Different Methods of RNA Preparation from Peripheral Blood for Nucleic Acid Amplification Assay

Background: Nucleic acid amplification assays (NAAs), such as polymerase chain reaction or loop-mediated isothermal amplification (LAMP), are used for disease diagnosis. Current nucleic acid isolation kits require several hours for completion of protocol including the complicated handling steps. Objective: In this study, a simple and cost-effective nucleic acid preparation method was developed, and its performance was compared with those of commercial kits. Materials and Methods: RNA was prepared using our method and three commercial RNA isolation kits. The RNA quantity and quality were evaluated using the NanoDrop spectrophotometer and Agilent 2100 bioanalyser. Reverse transcription LAMP (RT-LAMP) reactions were performed to determine the usability of the RNA preparation methods. Results: The concentrations of RNA extracted from blood samples by four different methods were sufficient for use in NAAs. The RNA integrity number was >7.0 when RNA was isolated using other RNA isolation kits but lower when prepared using our method. The RT-LAMP reaction was successfully performed when RNA was prepared using any of the methods. Conclusions: These results demonstrate that despite the lower purity and integrity of RNA, our RNA preparation protocol is simple and rapid and shows reasonable performance in RT-LAMP.

Does duration of cooking have an effect on PCR detection of meat for forensic purposes?

Although conventional Polymerase Chain Reaction (PCR) is one of the best methods used for meat identification for forensic purpose, some samples of cooked meat presented to Veterinary Research Institute for meat species identification have not responded in conventional PCR. Objective of this research was to exclude one of the possible reasons that would have caused this problem. To identify the effect of different cooking time on Deoxyribo Nucleic Acids (DNA) extraction and PCR, beef was used as the meat type, since very often the suspicious sample is claimed to be ‘beef’. Total of 18 samples of beef from 3 different commercial sources were used. Samples (n=6) from each source were cut into equal sizes and cooked separately to minimize contamination. They were cooked at 20 min, 40 min and 60 min cooking periods by adding equal amounts of commercially available products of turmeric powder, curry powder, chillie powder, salt powder and water. Samples were kept separately until DNA extraction. Forward and reverse primers were used for DNA amplification of bovine cytochrome b gene. The samples were subjected to DNA quantification by using the nanodrop spectrophotometer. Change in absorbance by DNA samples was used to quantify the DNA samples. The results of gel electrophoresis revealed that the samples were positive in all 3 cooking conditions with bands of ~ 272 bp equivalent compared to ladder and the positive control sample. Statistical analysis of DNA quantities revealed that even though the cooking time (up to 60 min) had no effect on the extracted DNA for species identification of beef samples as mentioned above, the DNA samples extracted from beef samples at 60 minutes resulted in high absorbance values indicating possible denaturation and fragmentation of DNA.

Enteric Infections in Young Children are Associated with Environmental Enteropathy and Impaired Growth.

To investigate the relationship between faecal contamination in child play spaces, enteric infections, environmental enteropathy (EE) and impaired growth among young children. A prospective cohort study was conducted of 203 children 6-30months of age in rural Bangladesh. Stool samples were analysed by quantitative PCR for Shigella, Enterotoxigenic Escherichia coli (ETEC), Campylobacter jejuni, Giardia intestinalis and Cryptosporidium spp. Four faecal markers of intestinal inflammation were also measured: alpha-1-antitrypsin, myeloperoxidase, neopterin and calprotectin. Child growth was measured at baseline and 9months after enrolment. E.coli was measured in soil in child play spaces. Forty-seven percent of study children had three or more enteric pathogens in their stool. Thirty five percent (71/203) of children had Shigella, 30% (61/203) had ETEC, 73% (148/203) had C.jejuni, 79% (160/203) had Giardia intestinalis and none had Cryptosporidium. Children with ETEC had significantly higher calprotectin concentrations (Coefficient: 1.35, 95% Confidence Interval [CI]: 1.005, 1.82). Children with Shigella had a significantly higher odds of being stunted at our 9-month follow-up (OR: 2.01, 95% CI: 1.02, 3.93). Children with Giardia intestinalis had significantly higher E.coli counts in the soil collected from their play spaces (OR: 1.23, 95% CI: 1.02, 1.48). Enteric infections were significantly associated with EE and impaired growth in rural Bangladesh. These findings provide further evidence to support the hypothesis that contaminated soil in child play spaces can lead to enteric infections, many of which are likely subclinical, resulting in EE and impaired growth in young children.

Q192R polymorphism in the PON1 gene and familial hypercholesterolemia in a Saudi population.

BACKGROUNDFamilial hypercholesterolemia (FH) is an autosomal dominant condition characterized by abnormal levels of low-density lipoprotein (LDL) in the blood. FH is a risk factor for atherosclerosis and cardiovascular disease. The relationship between the paraoxonase 1 (PON1) gene, atherosclerosis and coronary artery disease has not been studied in Saudi patients.OBJECTIVETo investigate the genetic associations of the Q192R polymorphism in the PON1 gene with FH in Saudi patients.DESIGNCase-control study.SETTINGTertiary care center, Riyadh.METHODSTwo hundred Saudi patients were enrolled in this study, including 100 patients with FH and 100 healthy controls, during the period from January 2012 to March 2013. Serum was separated from coagulated blood (3 mL) and used for analysis of lipid profiles. Genomic DNA was isolated from anticoagulant-treated blood (2 mL). Genotyping for the Q192R polymorphism was performed by polymerase chain reaction-restriction fragment length polymorphism analysis, followed by 3% agarose gel electrophoresis.MAIN OUTCOME MEASUREThe strength of association between the Q192R polymorphism and FH in the Saudi population.RESULTSWe confirmed that QR versus QQ (odds ratio [OR]: 1.55; 95% confidence interval [CI]: 1.05–3.43; P=.03), QR+RR versus QQ (OR: 1.98; 95% CI: 1.13–3.49; P=.01), and R versus Q (OR: 1.68; 95% CI: 1.09–2.59; P=.01) in the Q192R polymorphism were associated with FH in the Saudi population.CONCLUSIONIn conclusion, the Q192R polymorphism in the PON1 gene is associated with FH in the Saudi population. Our results confirmed that the R allele, QR, and dominant model genotypes were associated with FH.LIMITATIONOnly a single variant (Q192R) was analyzed, and the medical and family histories of the patients were not known.

The levels of yield and purity of genomic DNA from five tomato cultivars subjected to two DNA extraction techniques

Isolation of good quality genomic DNA from different plant materials is an important prerequisite for many molecular techniques related to both basic and applied research in the areas of plant molecular biology, crop improvement, biodiversity studies and conservation of genetic materials. Therefore, the need to extract reasonably pure DNA of both good quality and quantity for the downstream successful DNA-based diagnostic techniques is required. In this study, yield and purity of the extracted DNAs, using Dellaporta et al. (1983) and the use of Zymo Research Plant/Seed DNA MiniPrep Kit (ZR kit), were quantified with Nanodrop spectrophotometer and on agarose gel after polymerase chain reaction (PCR) amplification. The results of the DNA yield and purity were in the range of 24.42 to 1430.52 ng/μl and 1.11 to 2.00, respectively. The outstanding DNA yield in Kerewa cultivar in yield (1430.52 ng/μl) and purity (1.99) makes it appropriate for downstream DNA-based diagnostic techniques in biotechnology research. On agarose gel DNA bands appeared to be compact with negligible smearing with Dellaporta et al. method indicating good quality of DNA. The results demonstrated that good yield of very pure, intact, high quality genomic DNA could be obtained from tomato leaves using the method of Dellaporta et al. Keywords : DNA, extraction, purity, tomato, yield.

Abstract 1477: Melatonin regulates the tumor suppressor miR-148a-3p involved in angiogenesis and metastasis of breast cancer

Abstract Women with breast cancer has the tumor progression and angiogenesis-induced metastasis as the main cause of death. MicroRNAs (miRNAs) are small noncoding mRNA molecules that play an important role in gene regulation and once deregulated, these molecules may be involved with the progression of different human tumor types, including breast cancer. These miRNAs play an oncostatic role on tumor suppressor genes and regulate the process of angiogenesis, tumor growth and metastasis. So as new possible adjuvant treatment against breast cancer our group have shown melatonin, that is a hormone secreted by the pineal gland, by exhibiting several anti-tumor and antiangiogenic effects. Therefore, the aim of this study was to evaluate the potential therapeutic of melatonin on miRNAs regulation to verify breast cancer progression and potent tumor suppressor miR-148a-3p. In silico analysis was performed for selection of miRNAs involved in breast cancer. The MDA-MB-231 breast cancer cell line (metastatic negative estrogen receptor) was grown and separated in two different experimental conditions maintained for 24 hours: control group and melatonin-treated group (concentration of 1 mM). After this period the extraction of total RNA was performed (Qiagen®) and the total concentration of miRNAs of each sample was determined (NanoDrop Spectrophotometer 2000C - Thermo Scientific®). Differential expressions of these miRNAs was evaluate using miScript miRNA PCR-Array (Qiagen®) containing 84 miRNAs associated with breast cancer. The overexpression of miR-148a-3p in MDA-MB-231 cells was performed by bacterial cloning vector and the relative quantification of gene expression of their target IGF-1R and VEGF by real-time PCR. Moreover, the quantification of protein expression was performed by immunocytochemistry of IGF-1R and VEGF. Lastly, the migration and invasion cell assays were carried out to assess the potential capacity of these cells to undergo metastasis. The analysis of miRNAs MDA-MB-231 cell line by PCR-array showed 24 upregulated miRNAs and 8 miRNAs downregulated after the treatment with melatonin. Relative quantification shows that the melatonin treatment increases the gene expression of miR-148a-3p and decreases the gene and protein levels of IGF-1R and VEGF. The capability of migration and invasion of the breast cancer cells decreased after treatment with melatonin and overexpression of miR-148a-3p. Our results confirm the action of melatonin on the miR-148a-3p regulation known being involved in the progression of breast cancer. Thus the establishment of this therapeutic protocols can control these cellular events essential for the prognosis of patients with breast cancer. Citation Format: Debora Zuccari, Jéssica Zani Lacerda, Lívia Carvalho Ferreira, Beatriz Camargo Lopes, Andrés Felipe Aristizábal-Pachón, Marcio Chaim Bajgelman. Melatonin regulates the tumor suppressor miR-148a-3p involved in angiogenesis and metastasis of breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1477. doi:10.1158/1538-7445.AM2017-1477

Use of 16S rDNA Sequencing to Determine Procaryotic Diversity of a Remote Aviation Fuel-Polluted Lentic Ecosystem in Ibeno, Nigeria

Ibeno, the operational base of Mobil Producing Nigeria Unlimited, a subsidiary of ExxonMobil, Nigeria remains one of the most impacted communities by oil and gas activities in the Niger Delta region of Nigeria. Lotic and lentic systems in the region which residents rely on, receive petroleum hydrocarbon inputs almost daily due to oil spills and oily wastes discharges from operators and bunkering activities. This research was carried out to determine the prokaryotic diversity in a remote aviation fuel-polluted lentic ecosystem after 16 years of pollution using metagenomic approaches. DNA extraction from the water samples was carried out using MoBio DNA extraction Kits following the manufacturer’s instructions. Extracted DNA fragments were quantified using picogreen and by recording their UV absorption spectra using NanoDrop spectrophotometer. 16S rDNA sequencing was carried out on a Miseq Illumina sequencing platform and Quantitative Insight Into Microbial Ecology (QIIME) bioinformatics pipeline. Analyses revealed the dominance of bacterial and archaeal communities in both polluted and unpolluted water samples. The polluted sample had 93.83% bacterial and 3.43% archaeal population while the control sample revealed 58.05% bacterial and 39.69% archaeal population. Dominant bacterial phyla from the polluted samples were Proteobacteria, Firmicutes, Actinobacteria, Cyanobacteria, and Chloroflexi while dominant phyla in the unpolluted samples were Proteobacteria, Firmicutes and Actinobacteria. Dominant archaeal phyla from both polluted and unpolluted waters were Euryarchaeota and Crenarchaeota. The use of 16S rDNA metagenomic approach revealed a wide variety of bacterial and archaeal diversity from both polluted and control sites, thus revealing the true ecological status of both sites.

Effect of a Mushroom (Coriolus versicolor) Based Probiotic on the Expression of Toll-like Receptors and Signal Transduction in Goat Neutrophils

<p class="1Body">Neutrophils recognize and destroy pathogens through activation of the Toll like receptor (TLR) system as part of the inflammatory response of innate immunity. The expression and modulation of genes in the TLR signaling pathway in caprine blood neutrophils was investigated. Following initial screening for infection, goats (N=15) were assigned to three groups of five (n=5) individuals. Goats were drenched daily with 10 mL of powdered CorPet (Mycology labs Inc) soaked in hot (treatment I) or cold (treatment II) sterile filtered endotoxin free water, for a 4-week period. A control group of five age-matched goats received sterile water (treatment III). Blood was collected weekly and analyzed for packed cell volume and white blood cell differential counts. At weeks 1 and 4 neutrophils were isolated, using differential centrifugation and hypotonic lysis of red blood cells. The concentration and purity of total RNA isolated using Trizol was determined on a Nanodrop spectrophotometer. The RETROscript kit was used to synthesize cDNA. The expression of 84 genes in the human TLR signaling pathway RT<sup>2</sup> PCR Array was evaluated using real time PCR and the Livak method. The house keeping gene GAPDH was used to normalize the data. At week 1 untreated goats expressed 48 genes in the pathway. Goat neutrophils expressed 10 TLRs. Mushroom extracts modulated expression of and signaling by TLR. These results will help in the definition of the role of TLR expression in neutrophils and its contribution to goat innate immunity. Further this may aid in the design of therapeutics for goat health.</p>

Molecular detection of goat and sheep meat origin using mitochondrial cytochrome b gene

The present study was conducted to discriminate between sheep and goat species meat origin utilizing mitochondrial cytochrome b (Cyt b) gene fragment. A total of 46 ear tissue and meat samples were collected from different slaughterhouses and farms of Mymensingh and Rangpur districts. Genomic DNA was extracted using GeNet Bio DNA isolation kit and DNA concentration and purity was quantified by NanoDrop spectrophotometer. Two pairs species specific primer were used to amplify Cyt b gene fragments. Selected primers were highly conserved across the breed within a species and worked well with the species of indigenous goat and sheep resulting similar size of the amplicons 330 and 585 bp respectively. The duplex PCR condition would enable to detect adulteration from goat and sheep mixed samples which revealed by two precise bands (330 and 585 bp) in a single reaction. This study suggests an accurate molecular technique for identification of sheep and goat meat species origin and differentiates species present in adulterate meat samples. In conclusion, this DNA based marker could be used for prevention of fraudulent practice in slaughterhouse and chain shops in Bangladesh.Bang. J. Anim. Sci. 2016. 45 (2): 41-45

Comparison between Two Different DNA Extraction Techniques Taken from Buccal Swabs Suitable for Genetic Analyzer

This study aimed to validate two DNA extraction methods collected from buccal cells to gain a genomic DNA and to determine the effect of process time (from collection time to amplification). Total number of 116 buccal swab samples from unrelated healthy volunteers were subjected to DNA extraction by using two methods: modified (phenol chloroform) (Organic) and Prep Filer Forensic DNA Extraction kit. DNA was evaluated according to the: quantity, integrity and suitability for genetic analysis. The DNA yield and purity were measured by Nano drop Spectrophotometer. Organic method was modified from the classical method (addition Dithiothreitol to lysis buffer), The purity average values increased from 1.4 to 1.6 and it had the highest average concentration (73.3ng/µl) while the highest purity was observed in Prep Filer Forensic DNA Extraction kit with average (1.82). Delayed in sample processing effect both quantity and integrity of the extracted DNA. All samples were genetically analyzed at 16 STR with amelogenin (DNA markers) included in the Amp FlSTR® Identifiler™ PCR Amplification Kit panel from Applied Biosystems. Buccal cell swabs were very good source of genomic DNA especially for large population studies.

Normalization of gene expression measurement of tissue samples obtained by transurethral resection of bladder tumors.

BackgroundSample processing is a crucial step for all types of genomic studies. A major challenge for researchers is to understand and predict how RNA quality affects the identification of transcriptional differences (by introducing either false-positive or false-negative errors). Nanotechnologies help improve the quality and quantity control for gene expression studies.Patients and methodsThe study was performed on 14 tumor and matched normal pairs of tissue from patients with bladder urothelial carcinomas. We assessed the RNA quantity by using the NanoDrop spectrophotometer and the quality by nano-microfluidic capillary electrophoresis technology provided by Agilent 2100 Bioanalyzer. We evaluated the amplification status of three housekeeping genes and one small nuclear RNA gene using the ViiA 7 platform, with specific primers.ResultsEvery step of the sample handling protocol, which begins with sample harvest and ends with the data analysis, is of utmost importance due to the fact that it is time consuming, labor intensive, and highly expensive. High temperature of the surgical procedure does not affect the small nucleic acid sequences in comparison with the mRNA.ConclusionGene expression is clearly affected by the RNA quality, but less affected in the case of small nuclear RNAs. We proved that the high-temperature, highly invasive transurethral resection of bladder tumor procedure damages the tissue and affects the integrity of the RNA from biological specimens.

Single Lysis-Salting Out Method of Genomic DNA Extraction From Dried Blood Spots.

Dried blood spots (DBS) are an important form of bio-sampling and valuable approach for storing blood samples for genetic studies. This has necessitated in developing an effective protocol to isolate genomic DNA (gDNA) from DBS samples.In this study, we have elucidated a dependable and non-hazardous "single lysis-salting out" (SLSO) protocol of gDNA extraction from DBS and compared against the available commercial kits. For the purpose of this study, blood spots were collected on S&S 903 filter cards from 10 healthy volunteers and 30 patients with glutaric aciduria type I (GA-I). The gDNA was extracted from theseDBS samples by SLSO, QIAamp® gDNA Micro kit and innuPREP forensic kit methods. The quantity and quality of gDNA obtained from these methods were determined by measuring the absorbance using a Nanodrop spectrophotometer. The SLSO method showed four-fold and eight-fold increased yield of gDNA in healthy volunteers and patient samples, respectively, compared to commercial kits (p<0.0001). The protocol was also found to be cost efficient, reducing the per sample cost to almost half. The suitability of this method for genetic studies was confirmed by performing R402W genotyping by RFLP in GA-I patients. The genotyping results showed the presence of R402W mutation in 20% (6/30) of patients. The SLSO method was found to be inexpensive, non-hazardous and a suitable technique for isolating gDNA from DBS samples for genetic studies.

Ultraminiaturized assay for rapid, low cost detection and quantification of clinical and biochemical samples.

Herein, we report a simple, sensitive, rapid and low-cost ultraminiaturized assay technique for quantitative detection of 1μl of clinical or biochemical sample on a novel ultraminiaturized assay plate (UAP). UAP is prepared by making tiny cavities on a polypropylene sheet. As UAP cannot immobilize a biomolecule through absorption, we have activated the tiny cavities of UAP by 1-fluoro-2-nitro-4-azidobenzene in a photochemical reaction. Activated UAP (AUAP) can covalently immobilize any biomolecule having an active nucleophilic group such as amino group. Efficacy of AUAP is demonstrated by detecting human IgE, antibody of hepatitis C virus core antigen and oligonucleotides. Quantification is performed by capturing the image of the colored assay solution and digitally quantifying the image by color saturation without using costly NanoDrop spectrophotometer. Image - based detection of human IgE and an oligonucleotide shows an excellent correlation with absorbance - based assay (recorded in a NanoDrop spectrophotometer); it is validated by Pearson's product-moment correlation with correlation coefficient of r = 0.9545088 and r = 0.9947444 respectively. AUAP is further checked by detecting hepatitis C virus Ab where strong correlation of color saturation with absorbance with respect to concentration is observed. Ultraminiaturized assay successfully detects target oligonucleotides by perfectly hybridizing with their respective complementary oligonucleotide probes but not with a random oligonucleotide. Ultraminiaturized assay technique has substantially reduced the requirement of reagents by 100 times and assay timing by 50 times making it a potential alternative to conventional method.