Isolation of High-Quality RNA from Reaumuria soongorica, a Desert Plant Rich in Secondary Metabolites

Abstract

RNA isolation is a prerequisite for the study of the molecular mechanisms of stress tolerance in the desert plant Reaumuria soongorica, an extreme xeric semi-shrub. However, R. soongorica that contains high levels of secondary metabolites that co-precipitate with RNA, making RNA isolation difficult. Here the authors propose a new protocol suitable for isolating high-quality RNA from the leaves of R. soongorica. Based on a CTAB method described by Liu et al., the protocol has been improved as follows: the samples were ground with PVPP to effectively inhibit the oxidation of phenolics, contaminating DNA was removed with DNase I, and NaAc was used along with ethanol for precipitation to enhance the RNA yield and shorten the precipitation time. Gel electrophoresis and spectrophotometric analysis indicated that this isolation method provides RNA with no DNA contamination. Moreover, the yield (183.79 ± 40.36 μg/g) and quality were superior to those using the method of Liu et al., which yields RNA with significant DNA contamination at 126.30 ± 29.43 μg/g. Gene amplification showed that the RNA obtained using this protocol is suitable for use in downstream molecular procedures. This method was found to work equally well for isolating RNA from other desert plants. Thus, it is likely to be widely applicable.

Methodology An alternative cetyltrimethylammonium bromide-based protocol for RNA isolation from blackberry (Rubus L.)

“…The other detergents commonly used in lysing reagent formulation are SDS, urea and N-lauroyl sarcosine (Liao et al, 2004;Almarza et al, 2006;Rio et al, 2010;Wu et al, 2011). Methods involving CTAB, originally developed for pine tree tissue (Chang et al, 1993), is now prevalently chosen to extract nucleic acid from plant tissues, which are high in polysaccharides, polyphenols and other metabolites (Fort et al, 2008;Ghangal et al, 2009;Wang et al, 2011).…”

“…Thereafter, commercial kits have been developed through this method under different names such as 'TRIzol' (Invitrogen, USA), and 'RNeasy' (Qiagen, Germany). Unfortunately, most of these kits are not suitable for some plant species, especially those tissues rich in phenolic compounds, polysaccharides and other secondary metabolites, neither because of their inability to obtain RNA nor the negligible quality of RNA for downstream manipulations (Liu et al, 1998;Wang and Stegemann, 2010;Ghawana et al, 2011;Wang et al, 2011). Other conventional methods employing the hash lysate buffer such as sodium dodecyl sulfate (SDS), cetyltrimethyl ammonium bromide (CTAB), N-lauroyl sarcosine, and urea have also been widely used for RNA isolation in plant species (Liao et al, 2004;Almarza et al, 2006;Fort et al, 2008;Rio et al, 2010;Wu et al, 2011).…”

Isolation of High-Quality DNA from a Desert Plant Reaumuria soongorica

“…During the course of the RNA isolation, none of the kits were able to isolate any RNA from the leaves of R. soongorica (Wang et al 2011), so we did not try more other commercial DNA kits to isolate DNA, but attempted to use a improved CTAB method to extract DNA from the leaves of R. soongorica. When we carried out the modified CTAB-A method based on the classical Doyle and Doyle (1987) method, which consistently resulted in significant RNA contamination of the DNA samples (Fig.…”

“…Maxim, an extreme xeric semi-shrub of Tamaricaceae, is a constructive and dominant species of desert shrub vegetation (Liu et al 1982;Wang et al, 2011;Bai et al, 2008). It is distributed widely on a large area of sand wasteland (Fig.…”

Analysis of differentially expressed genes under UV-B radiation in the desert plant Reaumuria soongorica

“…Total RNA of each sample was isolated using a modified CTAB-based method [30]. The frozen fresh samples were fully grinded with liquid nitrogen, and100 mg of which was transferred to RNase-free Eppendorf tubes filled with 600 µl of pre-warmed CTAB extraction buffer containing 30 µl β-mercaptoethanol.…”