Difficulties in isolating total RNA FROM SPORULATING SOLVENTOGENIC CLOSTRIDIA

Kód výsledku v IS VaVaI

<a href="https://www.isvavai.cz/riv?ss=detail&h=RIV%2F60461373%3A22330%2F17%3A43913809" target="_blank" >RIV/60461373:22330/17:43913809 - isvavai.cz</a>

Výsledek na webu

<a href="http://www.icct.cz/AngiologyKlon-ICCT/media/system/ICCT2017-full_papers.pdf" target="_blank" >http://www.icct.cz/AngiologyKlon-ICCT/media/system/ICCT2017-full_papers.pdf</a>

DOI - Digital Object Identifier

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Jazyk výsledku

angličtina

Název v původním jazyce

Difficulties in isolating total RNA FROM SPORULATING SOLVENTOGENIC CLOSTRIDIA

Popis výsledku v původním jazyce

For a gene expression quantification using RNA-Seq and RT-qPCR, the proper purity and integrity of isolated total RNA is critical. The process of total RNA isolation using commercial kits is simple and user-friendly, but should be always adjusted to the type of target cells. When the target cells are sporulating bacteria, such as solventogenic clostridia, process of isolation faces multiple difficulties. The main problem is contamination with enzymes that cause the degradation of intact RNA, ribonucleases (RNases). External RNase contamination can happen during the whole process of isolation, as surfaces, instruments, solutions and hands of the researcher can be the source of RNases. Internal RNase contamination can happen after the step of cells lysis or earlier for the samples taken from the stationary phase of growth. The stationary phase of clostridia is characterized by high amount of spores, low viability of cell culture and lytic processes. These factors along with the high activity of RNases can cause the damage of RNA prior to the beginning of isolation. For the optimization of total RNA isolation from sporulating solventogenic clostridia two commercial kits (GenElute? Universal Total RNA Purification Kit, Sigma-Aldrich; High Pure RNA Isolation Kit, Roche) were compared. Quality, integrity and concentration of total RNA were measured using an Agilent 2100 Bioanalyzer (Agilent Technologies, Inc.) and micro-spectrophotometry (DS-11 FX+ Spectrophotometer (DeNovix, Inc.)). The results of our work show that, with the proper commercial spin column chromatography kit, it is possible to obtain total RNA from sporulating solventogenic clostridia with desired characteristics, proper for sequential RNA-Seq and RT-qPCR, despite the cell type difficulties.

Název v anglickém jazyce

Difficulties in isolating total RNA FROM SPORULATING SOLVENTOGENIC CLOSTRIDIA

Popis výsledku anglicky

For a gene expression quantification using RNA-Seq and RT-qPCR, the proper purity and integrity of isolated total RNA is critical. The process of total RNA isolation using commercial kits is simple and user-friendly, but should be always adjusted to the type of target cells. When the target cells are sporulating bacteria, such as solventogenic clostridia, process of isolation faces multiple difficulties. The main problem is contamination with enzymes that cause the degradation of intact RNA, ribonucleases (RNases). External RNase contamination can happen during the whole process of isolation, as surfaces, instruments, solutions and hands of the researcher can be the source of RNases. Internal RNase contamination can happen after the step of cells lysis or earlier for the samples taken from the stationary phase of growth. The stationary phase of clostridia is characterized by high amount of spores, low viability of cell culture and lytic processes. These factors along with the high activity of RNases can cause the damage of RNA prior to the beginning of isolation. For the optimization of total RNA isolation from sporulating solventogenic clostridia two commercial kits (GenElute? Universal Total RNA Purification Kit, Sigma-Aldrich; High Pure RNA Isolation Kit, Roche) were compared. Quality, integrity and concentration of total RNA were measured using an Agilent 2100 Bioanalyzer (Agilent Technologies, Inc.) and micro-spectrophotometry (DS-11 FX+ Spectrophotometer (DeNovix, Inc.)). The results of our work show that, with the proper commercial spin column chromatography kit, it is possible to obtain total RNA from sporulating solventogenic clostridia with desired characteristics, proper for sequential RNA-Seq and RT-qPCR, despite the cell type difficulties.

Druh

D - Stať ve sborníku

CEP obor

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OECD FORD obor

10606 - Microbiology

Projekt

<a href="/cs/project/GA17-00551S" target="_blank" >GA17-00551S: Vztah mezi efluxem butanolu a tolerancí k butanolu u klostridií</a><br>

Návaznosti

P - Projekt vyzkumu a vyvoje financovany z verejnych zdroju (s odkazem do CEP)

Rok uplatnění

2017

Kód důvěrnosti údajů

S - Úplné a pravdivé údaje o projektu nepodléhají ochraně podle zvláštních právních předpisů

Název statě ve sborníku

Proceedings of the 5th International Conference on Chemical Technology

ISBN

978-80-86238-65-4

ISSN

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e-ISSN

neuvedeno

Počet stran výsledku

5

Strana od-do

97-101

Název nakladatele

Česká společnost průmyslové chemie (ČSPCH)

Místo vydání

Praha

Místo konání akce

Mikulov

Datum konání akce

10. 4. 2017

Typ akce podle státní příslušnosti

WRD - Celosvětová akce

Kód UT WoS článku

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