Effect of pH and Ionic Strength on the Spectrophotometric Assessment of Nucleic Acid Purity

Wilfinger, William W.; Mackey, Karol; Chomczyński, Piotr

doi:10.2144/97223st01

Cited by 421 publications

(273 citation statements)

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“…Factors other than nucleic acid purity, such as high pH, can give a false high 260/280 ratio. 21 This could be an explanation as to why all samples may seem to have good purity.…”

Section: Discussionmentioning

confidence: 99%

A novel approach for HLA-A typing in formalin-fixed paraffin-embedded-derived DNA

et al. 2014

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The aim of this study was to establish a novel approach for human leukocyte antigen (HLA)-typing from formalin-fixed paraffin-embedded-derived DNA. HLAs can be a prognostic factor in cancer and have an extensive polymorphism. This polymorphism is predominantly restricted to exons, which encode the peptidebinding domain of the protein. Formalin-fixed paraffin-embedded material is routinely collected in the clinic and therefore a great source of DNA for genetic analyses. However, its low quality due to fragmentation and nucleotide changes has often created obstacles in designing genetic assays. In this study, we amplified the most polymorphic exons of the HLA-A gene, exons 2, 3, and 4, in 16 formalin-fixed paraffin-embedded samples 410 years old. These tissue samples belonged to patients already HLA-typed by peripheral blood samples at the routine laboratory. Acquired amplification products were used for sequencing, which provided enough information to establish an HLA allele. The same method was applied to DNA extracted from peripheral blood from a healthy volunteer with known HLA type. Of the samples, 14/16 (88%) were successfully typed, in one sample only one of the alleles could be determined, and in one sample no allele could be determined. The amplification of the most polymorphic exons of HLA-A was a successful alternative when DNA quality prevented positive results with previously described methods. The method is usable when an HLA type is needed but the patients are deceased and/or no whole blood samples can be collected. It has thus potential to be used in several fields such as the clinic, research, and forensic science.

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“…Factors other than nucleic acid purity, such as high pH, can give a false high 260/280 ratio. 21 This could be an explanation as to why all samples may seem to have good purity.…”

Section: Discussionmentioning

confidence: 99%

A novel approach for HLA-A typing in formalin-fixed paraffin-embedded-derived DNA

et al. 2014

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“…13 DNA Concentration (ug ml -1 ) =OD at 260 nm × 50 ug ml -1 × dilution factor. The DNA extract samples were stored at -20°C until being used.…”

Section: Dna Extractionmentioning

confidence: 99%

An investigation of type 3 secretion toxins encoding-genes of Pseudomonas aeruginosa isolates in a University Hospital in Egypt

Gawish¹

2013

Jmid

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Objective: Pseudomonas aeruginosa uses type 3 secretion system (T3SS) to directly inject four toxins into host cells. The aim of the study was to determine the prevalence of the toxins-encoding genes among P. aeruginosa isolates collected from nosocomial infections and environmental sources.Methods: Clonal relatedness of the studied P. aeruginosa isolates (n=85) was studied by RAPD typing. Multiplex PCR was performed on the 85 P. aeruginosa isolates (68 nosocomial isolates obtained from hospitalized patients present in different wards and departments, and 17 environmental isolates including 2 isolates from health-care worker hands from ICU) to detect the secretion toxins-encoding genes.Results: RAPD fingerprinting demonstrated that most strains were of distinct genotypes and determined the presence of 54 RAPD patterns. The prevalence of the genes among all isolates was as follows; exoT-100%, exoS-40%, exoY-83.5% and exoU-62.4%. No significant differences in the prevalence of these genes were observed between nosocomial and environmental isolates or between isolates cultured from different sites of infection. The part of P. aeruginosa strains harboring either exoS (37.6%) or exoU (60%) gene was significantly higher (P<0.001) than that contained both genes (2.4%). Conclusion:ExoT was present in all isolates and exoS, exoY and exoU are variable traits with exoU gene was the third in prevalence after exoT and exoY. No significant differences in exoS, exoY and exoU prevalence were observed between nosocomial and environmental isolates or between isolates from different sites of infection. ExoS and exoU genes were mutually exclusive that almost all isolates contain either exoS without exoU or exoU without exoS. Yöntemler: Çalışılan P. aeruginosa izolatlarının klonal yakınlığı RAPD tiplendirme metodu ile bakılmıştır. Sekretuar toksin kodlayan genleri saptamak için 85 P. aeruginosa izolatında (farklı klinik ve bölümlerde yatan hastalardan izole edilen 68 hastane kaynaklı izolatı ve ikisi yoğun bakım ünitesindeki sağlık çalışanının ellerinden izole edilen 17 çevresel izolat) multiplex PCR çalışıldı.Bulgular: RAPD fingerprint analizi ile varlığı tespit edilen 54 RAPD paterninin varlığı suşların çoğunun uzak genotiplere sahip olduğunu gösterdi. Tüm izolatlarda saptanan genlerin prevalansı sırasıyla exoT (% 100), exoS (% 40), exoY (% 83,5) ve exoU (% 62,4) şeklindeydi. Farklı enfeksiyon bölgelerinden üretilen izolatlar arasında yada hastane ve çevresel kaynaklardan elde edilen izolatlar arasında bu genlerin prevalansı açısından anlamlı bir fark yoktu. P. aeruginosa suşla-rından exoS (% 37,6) veya exoU (% 60) genlerinden birini taşıyanlar her iki geni taşıyanlardan (% 2,4) anlamlı olarak daha fazlaydı (p<0.001). Sonuç:ExoT tüm izolatlarda bulunurken exoS, exoY ve exoU genlerinin varlığı değişkenlik gösteriyordu. ExoU geni exoT ve exoY geninden sonra üçüncü sıklıkta görülmekteydi. exoS, ExoY ve exoU görülme prevalansı hastane kaynaklı ve çev-re kaynaklı suşlar arasında veya değişik bölge veya enfeksiyonlar arasında an...

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“…Concentration of RNA sample was measured 44 ug mL −1 A 260 (Wilfinger et al, 1997). The ratio of the reading at (A 260 /A 280 ) provides am estimate of the purity of RNA.…”

Section: Spectrophotometric Quantification Of Rnamentioning

confidence: 99%

Factors Those Up Regulate <i>Klotho</i> and Glutathione Peroxidase-1 Gene Expression Improve Renal Function in Rats with Acute Renal Failure

Elgendey¹,

Hemeda²,

Sosa³

et al. 2015

American Journal of Biochemistry and Biotechnology

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Acute Renal Failure (ARF) has traditionally been defined as the abrupt and progressive loss of kidney functions resulting in the retention of urea and other nitrogenous waste products associated with interstitial inflammation, tubular injury and increasing Tumor Necrosis Factor (TNF). Mortality in patients with ARF remains high >50% in severely ill patients. Klotho gene is a new anti-aging gene. Genetic mutation of klotho gene causes multiple premature aging-like phenotypes and shortens lifespan. Klotho gene is highly expressed in the kidney and a soluble form of klotho functions as an endocrine substance that exerts multiple actions including the modulation of renal solute transport and the protection of the kidney. This study aimed to clarify the pre treatment and/or post treatment effect of vitamin E as an antioxidant on kidney functions, klotho gene expression and glutathione peroxidase-1 gene expression among rats with acute renal failure. Using glycerol as oxidative stress factor to cause acute renal failure and Real time PCR for assessment of gene expression of target gene in the control and treated groups. Our results demonstrated that the vitamin E (α tocopherol) as antioxidant factor decreased the kidney injuries as pre renal failure administer and improve kidney function as post renal failure administer. Those effects were through up regulating the Klotho as anti aging gene and the Glutathione Peroxidase (GPx-1) as antioxidant gene expression in the kidney tissue. We concluded that factors those up regulate the klotho gene expression can use as protective factors against kidney injuries and to improve kidney function in renal failure.

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Effect of pH and Ionic Strength on the Spectrophotometric Assessment of Nucleic Acid Purity

Cited by 421 publications

References 1 publication

A novel approach for HLA-A typing in formalin-fixed paraffin-embedded-derived DNA

A novel approach for HLA-A typing in formalin-fixed paraffin-embedded-derived DNA

An investigation of type 3 secretion toxins encoding-genes of Pseudomonas aeruginosa isolates in a University Hospital in Egypt

Factors Those Up Regulate <i>Klotho</i> and Glutathione Peroxidase-1 Gene Expression Improve Renal Function in Rats with Acute Renal Failure

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