Isolation of High Quality RNA from Cereal Seeds Containing High Levels of Starch

Wang, Guifeng; Gang, Wang; Zhang, Xiaowei; Wang, Fang; Song, Rentao

doi:10.1002/pca.1337

Cited by 113 publications

(90 citation statements)

References 13 publications

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“…Considering that high salt lysis buffer could reduce the impact of polysaccharides, starches and other metabolites on nucleic acids (Fang et al, 1992;Wang et al, 2012), CTAB was used as the main cell disruption and RNase inactivation reagent instead of SDS. Significant modifications of the method presented here included more CTAB (3%), and higher Trisbuffer concentration (200 mM) to increase the buffer capacity.…”

Section: Discussionmentioning

confidence: 99%

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

Chen

Yu²,

Wang³

et al. 2012

Genet. Mol. Res.

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ABSTRACT. Isolation of high-quality RNA free of contaminants, such as polyphenols, proteins, plant secondary metabolites, and genomic DNA from plant tissues, is usually a challenging but crucial step for molecular analysis. We developed a novel protocol based on the cetyltrimethylammonium bromide method to isolate high-quality RNA from blackberry plant tissues, especially fruits. Most DNA was removed when acetic acid was utilized, before RNA precipitation. Thus, lithium chloride, a reagent widely used for RNA purification, was not needed. The isolation time was shortened to less than 3 h. The RNA was quite pure, with little DNA contamination. The quality of the RNA was assessed by spectrophotometric readings and electrophoresis on agarose gels. It was good enough for downstream enzymatic reactions, such as reverse transcription-PCR, cloning and real-time PCR assay. The method yielded an amount of total RNA comparable to previously described protocols.

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Section: Discussionmentioning

confidence: 99%

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

Chen

Yu²,

Wang³

et al. 2012

Genet. Mol. Res.

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“…The flashfrozen tissues were pulverized, and RNA was extracted (Wang et al, 2012). The isolated RNA preparations were treated with DNase (Ambion), and 8 mg of RNA was reverse transcribed using Double Primed RNA to cDNA EcoDry Premix (Clontech).…”

Section: Rna Extraction and Quantitative Pcrmentioning

confidence: 99%

Spatial Mapping and Profiling of Metabolite Distributions during Germination

et al. 2017

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Germination is a highly complex process by which seeds begin to develop and establish themselves as viable organisms. In this study, we utilize a combination of gas chromatography-mass spectrometry, liquid chromatography-fluorescence, and mass spectrometry imaging approaches to profile and visualize the metabolic distributions of germinating seeds from two different inbreds of maize (Zea mays) seeds, B73 and Mo17. Gas chromatography and liquid chromatography analyses demonstrate that the two inbreds are highly differentiated in their metabolite profiles throughout the course of germination, especially with regard to amino acids, sugar alcohols, and small organic acids. Crude dissection of the seed followed by gas chromatography-mass spectrometry analysis of polar metabolites also revealed that many compounds were highly sequestered among the various seed tissue types. To further localize compounds, matrix-assisted laser desorption/ionization mass spectrometry imaging was utilized to visualize compounds in fine detail in their native environments over the course of germination. Most notably, the fatty acyl chain-dependent differential localization of phospholipids and triacylglycerols was observed within the embryo and radicle, showing correlation with the heterogeneous distribution of fatty acids. Other interesting observations include unusual localization of ceramides on the endosperm/scutellum boundary and subcellular localization of ferulate in the aleurone.Plants use seeds as the propagule to ensure reproduction to the next generation, and over the past 10,000 years, human civilizations have established agricultural practices to ensure a seed-based food supply (Larson et al., 2014). Therefore, deciphering the processes that enable seeds to perform their biological functions is of importance in understanding how plants are propagated and also is of practical importance to improve agriculture. Seeds are designed to survive long periods of dormancy in a relatively dry state, and the process of germination is initiated by the imbibition of water. During this germination process, many metabolic changes occur, most of which are associated with the catabolism of seed storage products (proteins, polysaccharides, and lipids) into metabolically usable, simpler chemical forms that are either used as precursors to assemble the growing seedling or are oxidized via energy-producing biochemical pathways to thermodynamically support growth (Bewley, 1997(Bewley, , 2001.The specific metabolic processes that support seed germination are somewhat dependent on the taxonomic clade of the plant, which determines seed tissue/ organ organization and the nature of the seed storage compounds. Specifically, the seeds of the Poaceae family of monocots are characterized by a starch-and protein-filled endosperm, and their catabolism by digestive enzymes produced in the outermost layer of the endosperm (i.e. the aleurone) provides the carbonbased and nitrogenous precursors for the growth of the embryo, which is composed of the embryonic ax...

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“…In fact, CTAB-based methods have recently been used for nucleic acid isolation from polysaccharide-rich plants, and in particular, these methods have been used for RNA isolation (Thanh et al, 2009;Abbasi et al, 2010;Wang et al, 2012). Both A 260 /A 230 and A 260 /A 280 ratios were more than 2 suggesting that the RNA was of high purity and free of polysaccharide/polyphenol and protein contamination, respectively (Table 1) (Wang et al, 2005;Yang et al, 2011 Table 1.…”

Section: Resultsmentioning

confidence: 99%

“…The previous plant RNA isolation protocols, including acidic guanidinium thiocyanate-based, SDS/phenol and hot borate methods, have proved unsuitable to isolate RNA when applied to tissues containing high levels of secondary metabolites (Xu et al, 2010;Wang et al, 2012). For RNA isolation from lotus, the large amounts of secondary metabolites mentioned above make it particularly difficult.…”

Section: Introductionmentioning

confidence: 99%

“…These medicinal properties of lotus have attracted worldwide interest in pharmaceutical and genetic diversity research; however, investigation of the functional genes in this plant is limited. The limitation may be partially due to the presence of polysaccharides, polyphenols, flavonoids, and alkaloids in its tissues, which severely interfere with DNA, RNA and protein purification (Lönneborg and Jensen, 2000;Wang et al, 2012).…”

Section: Introductionmentioning

confidence: 99%

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Short Communication Efficient isolation of high-quality RNA from lotus Nelumbo nucifera ssp nucifera tissues

Zhang¹,

Hao²,

Liang³

et al. 2013

Genet. Mol. Res.

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ABSTRACT. Nelumbo nucifera is widely used as food, as an ornamental, in medicine, and as packing material; it is also reported to have anti-HIV effects and antioxidant capacity. We sought an improved method for extracting high-quality total RNA from different tissues of N. nucifera. Four methods for RNA extraction were assessed for their ability to recover high-quality RNA applicable for evaluation of polyphenol oxidase (PPO) gene expression profiles. The recovery and quality of the RNA obtained from five different tissues by the best CTAB-LiCl method were evaluated through UV light absorbance. Both A 260 /A 280 and A 260 /A 230 absorbance ratios were more than 2.0; the yield ranged from 59.87 to 163.75 μg/g fresh weight. The brightness of the 28S band was approximately twice that of 18S; the latter was also considered as high-quality RNA. The PPO gene fragment (606 bp) was successfully amplified by RT-PCR, demonstrating the integrity of the isolated RNA. The relative expression levels of the PPO gene based on RT-PCR in five tissues of lotus were: rhizome buds (2.66), young leaves (2.42), fresh cut rhizome (2.02), petals (1.80), and petiole (1.65), using housekeeping gene β-actin as an internal control. We concluded that the total RNA isolated by this protocol is of sufficient quality for molecular applications.

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Isolation of High Quality RNA from Cereal Seeds Containing High Levels of Starch

Abstract: The study has described an easy, efficient and highly reproducible method for RNA isolation from various cereal seeds.

Cited by 113 publications

References 13 publications

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

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

Spatial Mapping and Profiling of Metabolite Distributions during Germination

Short Communication Efficient isolation of high-quality RNA from lotus Nelumbo nucifera ssp nucifera tissues

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