Acta Univ. Agric. Silvic. Mendelianae Brun. 2008, 56(4), 219-222 | DOI: 10.11118/actaun200856040219
Ověření stability RNA pomocí reverzní transkripce a PCR v reálném čase
- Ústav morfologie, fyziologie a genetiky zvířat, Mendelova zemědělská a lesnická univerzita v Brně, Zemědělská 1, 613 00 Brno, Česká republika
Cílem studie bylo zjistit stabilitu izolované RNA v různých laboratorních podmínkách. Studie byla zaměřena na možné inhibiční vlastnosti vody ošetřené diethylpyrocarbonátem (DEPC), dále byl zkoumán vliv času (1, 2, 4, 8, 16, 32 a 64 hodin inkubace) a teploty (20 °C, 4 °C, -20 °C a -80 °C) na degradaci izolované RNA. Inkubované vzorky byly následně převedeny pomocí reverzní transkripce na cDNA. Její množství bylo analyzováno pomocí polymerázové řetězové reakce v reálném čase (real-time PCR). Dále byl zkoumán pouze vliv teploty inkubace nebo elučního roztoku na degradaci získané RNA. Statistické zhodnocení výsledků bylo provedeno metodou analýzy variance (ANOVA) a nebyl zjištěn statisticky významný rozdíl mezi skupinami vzorků, respektive nedocházelo k významné degradaci RNA mezi pozorovanými skupinami vzorků. Zejména je důležité zjištění, že není významný rozdíl degradace RNA uchovávané ve vodě ošetřené DEPC a v RNáz prosté vodě. Výsledky této práce jsou použitelné pro veškeré molekulární aplikace, které pracují na bázi RNA.
RNA, RNásy, diethylpyrocarbonát, real-time PCR
Determination of RNA stability using reverse transcription real-time PCR
The aim of this study was to verify an effect of RNA storage in different laboratory conditions. Especially, this work was focused on the importance of using diethylpyrocarbonate (DEPC) treated water for storage of isolated RNA. The effect of storage of RNA samples in different temperatures was monitored according to various times as well. Isolated RNA was incubated at 20 °C, 4 °C, -20 °C and -80 °C, whereas the temperature -80 °C was used as a control. After incubation only mRNA was converted to cDNA by reverse transcription. The polymerase chain reaction in real time (real-time PCR) was used for a measurement of RNA degradation. No statistically significant interactions were found between RNA treatment conditions if analysis of variance (ANOVA) model was applied. The result showed that storage of isolated RNA in water treated with DEPC is not necessary. This approach prevents possible inhibition downstream reaction caused by DEPC. The results of this study can be used in all molecular applications based on RNA.
Keywords: RNA, RNases, diethylpyrocarbonate, real-time RT-PCR
Grants and funding:
Tato práce byla podpořena Grantovou agenturou České republiky, projekty č. 523/06/1302 a 523/03/H076.
Received: February 21, 2008; Published: November 8, 2014 Show citation
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