Wyniki wyszukiwania - Biblioteka Nauki

1

DNA vs RNA based studies of nitrogen removal bacteria genes via qPCR

100%

Banach-Wiśniewska A. , Gamoń F. , Ziembińska-Buczyńska A.

Archives of Environmental Protection

|

2021

|

tom Vol. 47, no. 1

19--25

EN

Improvements in water quality requires the removal of nitrogen compounds from wastewater. The most promising and cost-effective methods for this purpose are biological ones based on activated sludge microorganisms such as nitrifiers, denitrifiers, and anammox bacteria. Due to the most of the nitrogen removal bacteria are uncultivable in a laboratory, the application of the molecular tools is required to investigate microorganisms involved in the nitrogen removal. In case of this study for the analysis of relative genes abundance of nitrogen removal bacteria, quantitative PCR (qPCR) based on bacterial DNA and qPCR preceded by reverse transcription (RT-qPCR) based on bacterial mRNA as a template, were used with specific bacterial functional genes (amoA, nrxA, nirS, nirK, hzo). Samples from four anammox sequencing batch reactors (SBRs) were analyzed, while the nitrogen removal process and bacteria growth were supported by biomass immobilization and nanoparticles addition. There were statistically significant differences between results obtained in the case of mRNA and DNA (p<0.05). Statistically significant positive correlations were found between results obtained with those two approaches. In case of mRNA analysis, positive results were obtained only for hzo, amoA and partly for nirS genes, despite additional purification and removal of inhibitors from samples prior to reaction.

PL

Z uwagi na to, że większości bakterii przemian związków azotowych nie można wyizolować w postaci czystych kultur, do ich zbadania konieczne jest zastosowanie metod biologii molekularnej. Jedną z najczęściej stosowanych w tym celu jest ilościowa reakcja łańcuchowa polimerazy (ang. Quantitative Polimerase Chain Reaction, qPCR). Celem eksperymentu było porównanie wyników analizy wybranych genów funkcyjnych bakterii przemian związków azotowych przy pomocy metody qPCR wykonanej na matrycy DNA i RNA (po odwrotnej transkrypcji). Względna liczebności genów funkcyjnych analizowana była z zastosowaniem metody qPCR (na matrycy DNA) oraz RT-qPCR (ang. Reverse Transcription-qPCR) (na matrycy RNA). Analizę przeprowadzono w oparciu o geny: amoA, nrxA, nirS, nirK i hzo. Próbki osadu czynnego pobrano z czterech sekwencyjnych reaktorów porcjowych, w których proces usuwania azotu i wzrostu bakterii wspomagano za pomocą immobilizacji biomasy i dodatkiem nanocząstek. Wykazano statystycznie istotne różnice między wynikami uzyskanymi w przypadku badań mRNA i badań opartych na DNA (p<0,05). Wyniki uzyskane za pomocą zastosowanych narzędzi biologii molekularnej (qPCR, RT-qPCR) były skorelowane pozytywnie. W przypadku analizy opartej na mRNA pozytywne wyniki uzyskano tylko dla genów hzo, amoA i częściowo dla genów nirS, pomimo dodatkowego oczyszczania i usuwania inhibitorów z próbek przed reakcją. Należy podkreślić, że w zależności od matrycy zastosowanej w qPCR (bakteryjne DNA lub cDNA zsyntetyzowane z bakteryjnego mRNA w procesie odwrotnej transkrypcji) uzyskane wyniki mogą wskazywać na różne informacje naukowe. Pomimo znaczących różnic pomiędzy wynikami otrzymanymi za pomocą dwóch metod, obliczone współczynniki korelacji Spearmana wskazują na wzajemne powiązanie pomiędzy otrzymanymi wynikami oraz powiązania ekologiczne pomiędzy bakteryjnymi genami przemian związków azotowych.

2

Real-Time PCR w nauce i diagnostyce - możliwości aplikacyjne, analiza i interpretacja wyników

100%

Hołysz M.

Laboratorium - Przegląd Ogólnopolski

|

2014

|

tom nr 9-10

26--32

PL

Technika Real-Time PCR jest jedną z podstawowych technik molekularnych wykorzystywanych zarówno w laboratoriach naukowych, jak i w laboratoriach diagnostycznych. Technika ta z roku na rok zdobywa coraz większą grupę użytkowników, rozszerzają się możliwości aplikacyjne oraz zakres badań wykonywanych w oparciu o PCR w czasie rzeczywistym. Cele niniejszego artykułu to: prezentacja możliwości zastosowania techniki Real-Time PCR w badaniach naukowych i diagnostyce (w szczególności w diagnostyce klinicznej), podstaw teoretycznych poszczególnych aplikacji, a także zasad analizy i interpretacji wyników.

EN

Technique Real-Time PCR is one of the basic molecular techniques used both in laboratories and in diagnostic laboratories. This technique each year gaining more and more a group of users, expand application capabilities and the scope of the tests performed on the basis of real-time PCR. The purpose of this article is to present the possibility of using techniques Real-Time PCR in research and diagnostics (in particular clinical diagnosis), the theoretical basis of each application, as well as the principles of analysis and interpretation of results.

3

Molecular cloning, expression and characterization of Bmserpin-2 gene from Bombyx mori

99%

Pan Y. , Xia H. , Lü P. , Chen K. , Yao Q. , Chen H. , Gao L. , He Y. , Wang L.

Acta Biochimica Polonica

|

2009

|

tom 56

|

nr 4

671-677

EN

Serpins are a broadly distributed family of protease inhibitors. In this study, the gene encoding Bombyx mori serpin-2 (Bmserpin-2) was cloned and expressed in E. coli. The Bmserpin-2 cDNA contains a 1125 bp open reading frame (ORF). The deduced protein has 374 amino-acid residues, contains a conserved SERPIN domain and shares extensive homology with other invertebrate serpins. RT-PCR analysis showed that Bmserpin-2 was expressed in all developmental stages of B. mori larvae and various larval tissues. Subcellular localization analysis indicated that Bmserpin-2 protein was located in the cytoplasm. Interestingly, real-time quantitative PCR revealed that the expression of Bmserpin-2 in the midgut of susceptible B. mori strain 306 significantly increased at 72 hours post inoculation (hpi) when infected with BmNPV. However, there was no significant increase of the Bmserpin-2 expression in resistant strain NB infected with BmNPV. Thus, our data indicates that Bmserpin-2 may be involved in B. mori antiviral response.

4

Feeding state and age dependent changes in melanin-concentrating hormone expression in the hypothalamus of broiler chickens

85%

Simon Á. , Németh J. , Jávor A. , Komlósi I. , Bai P. , Oláh J. , Juhász B. , Kiss R. , Szilvássy Z. , Czeglédi L.

Acta Biochimica Polonica

|

2018

|

tom 65

|

nr 2

251-258

EN

We aimed to quantify the gene expression changes of the potent orexigenic melanin-concentrating hormone (MCH) in chicken (Gallus gallus) hypothalamus with quantitative real-time polymerase chain reaction (qPCR), and for the first time determine peptide concentrations with a novel radioimmunoassay (RIA) under different feeding status. Three different experimental conditions, namely ad libitum feeding; fasting for 24 h; fasting for 24 h and then refeeding for 2 h, were applied to study changes of the aforementioned target and its receptor (MCHR4) gene expression under different nutritional status. The relative changes of MCH and MCHR4 were also studied from 7 to 35 days of age. Expression of PMCH and MCHR4 along the gastrointestinal tract (GIT) was also investigated. We found that expression of both targets was significant in the hypothalamus, while only weak expression was detected along the GIT. Different nutritional states did not affect the PMCH and MCHR4 mRNA levels. However, fasting for 24 h had significantly increased the MCH-like immunoreactivity by 25.65%. Fasting for 24 h and then refeeding for 2 h had further significantly increased the MCH peptide concentration by 32.51%, as compared to the ad libitum state. A decreasing trend with age was observable for both, the PMCH and MCHR4 mRNA levels, and also for the MCH-like immunoreactivity. Correlation analysis did not result in a significant correlation between MCH peptide concentration and abdominal fat mass in ad libitum fed birds. In conclusion, MCH peptide concentration altered in response to 24 h fasting, which indicated that this peptide may take part in feed intake regulation of broiler chickens.

5

Different MACS sorting strategies for the enrichment of Lin– (CD34+ CD45–) hematopoietic progenitor cells: preliminary study

85%

Vašíček J. , Baláži A. , Parkányi V. , Bauer M.

Annales Universitatis Paedagogicae Cracoviensis Studia Naturae

|

2018

EN

Magnetic-activated cell sorting (MACS) has become a standard method for the isolation of hematopoietic stem/progenitor cells (HSC/HPC) in human or mouse model using CD34 antibodies. However, at the present there is no useable CD34 antibody that could be successfully used for the selection of rabbit HSC/HPC. Therefore, the aim of this preliminary study was to remove all mature cells (CD45+) from the heterogeneous mixture of rabbit peripheral blood and bone marrow mononuclear cells (PBMCs and BMMCs) in order to enrich these cell populations for the CD34+ cells. Briefly, cells were incubated with a CD45 antibody and proper magnetic microbeads. Three different MACS sorting strategies were used in the experiment that differed mainly in the sample loading rate and the number of used magnetic columns. Control (unsorted) and sorted cells were assessed for the sorting efficiency (% of double positive cells for CD45 and Labelling Check Reagent – LCR) by flow cytometry and for the relative expression of CD34 antigen by qPCR. According to flow cytometry, Depl025 mode showed the best sorting efficiency in terms of the lowest percentages of CD45+LCR+ cells for rabbit PBMCs as well as BMMCs. qPCR analysis confirmed this mode as the best in terms of the relative CD34 expression for rabbit PBMCs. However, higher relative expression of CD34 in BMMCs was obtained by other mode – Posselds. In conclusion, this study demonstrates a possible enrichment of rabbit (CD34+) HSC/HPC by the magnetic depletion of mature hematopoietic (CD45+) cells.

6

Soil microbiomes of reclaimed and abandoned mines of the Yamal region

71%

Pershina E. , Ivanova E. , Kimeklis A. , Zverev A. , Kichko A. , Aksenova T. , Andronov E. , Abakumov E. , Pershina E. , Ivanova E. , Kimeklis A. , Zverev A. , Kichko A. , Aksenova T. , Andronov E. , Abakumov E.

|