A robust plant RNA isolation method suitable for Affymetrix GeneChip analysis and quantitative real-time RT-PCR

Bilgin, Damla D.; DeLucia, Evan H.; Clough, Steven J.

doi:10.1038/nprot.2008.249

Cited by 64 publications

(49 citation statements)

References 16 publications

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“…RNA was isolated according to previous methods (Bilgin et al, 2009). The first-strand cDNA was synthesized from 2 mg total RNA, and the product was used for qRT-PCR on a real-time PCR detection system according to manufacturer's instructions (Bio-Rad CFX96).…”

Section: Rna Extraction and Qrt-pcrmentioning

confidence: 99%

DWARF AND LOW-TILLERING Acts as a Direct Downstream Target of a GSK3/SHAGGY-Like Kinase to Mediate Brassinosteroid Responses in Rice

et al. 2012

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In Arabidopsis thaliana, the GSK3/SHAGGY-like kinase BRASSINOSTEROID-INSENSITIVE2 (BIN2) plays a critical role in the brassinosteroid (BR) signaling pathway by negatively regulating the activities of bri1-EMS-SUPPRESSOR1/BRASSINAZOLE-RESISTANT1 family transcription factors that regulate the expression of downstream BR-responsive genes. In this study, we analyzed the function of a rice (Oryza sativa) GSK3/SHAGGY-like kinase (GSK2), which is one of the orthologs of BIN2. Overexpression of GSK2 (Go) led to plants with typical BR loss-of-function phenotypes, and suppression of GSK2 resulted in enhanced BR signaling phenotypes. DWARF AND LOW-TILLERING (DLT) is a positive regulator that mediates several BR responses in rice. Suppression of DLT can enhance the phenotypes of BR receptor mutant d61-1, and overexpression of DLT obviously suppressed the BR loss-of-function phenotypes of both d61-1 and Go, suggesting that DLT functions downstream of GSK2 to modulate BR responses. Indeed, GSK2 can interact with DLT and phosphorylate DLT. Moreover, brassinolide treatment can induce the dephosphorylation of DLT, leading to the accumulation of dephosphorylated DLT protein. In GSK2 transgenic plants, the DLT phosphorylation level is dictated by the GSK2 level. These results demonstrate that DLT is a GSK2 substrate, further reinforcing that the BIN2/GSK2 kinase has multiple substrates that carry out various BR responses.

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Section: Rna Extraction and Qrt-pcrmentioning

confidence: 99%

DWARF AND LOW-TILLERING Acts as a Direct Downstream Target of a GSK3/SHAGGY-Like Kinase to Mediate Brassinosteroid Responses in Rice

et al. 2012

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“…Precipitation of RNA using lithium chloride is not suitable for in vitro translation or reverse transcription based experiments as chloride ions will inhibit protein synthesis and DNA polymerase. While using TRIZOL reagent for RNA extraction, Bilgin et al (2009) had noticed the presence of organic contaminants in the isolated RNA. The objective of this study was to develop a simple, reliable RNA extraction protocol suitable for a variety of tropical medicinal plants, which meet the quality parameters for further downstream processes.…”

Section: Introductionmentioning

confidence: 99%

Simple and efficient method for functional RNA extraction from tropical medicinal plants rich in secondary metabolites

George¹

2018

Trop. Plant Res.

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Isolation of high-quality functional RNA is prerequisite to facilitate any study related to gene expression and also for downstream applications such as reverse transcription-polymerase chain reaction (RT-PCR), suppression subtractive hybridization (SSH) library construction, differential display (DD), real-time PCR and northern hybridization of medicinal plants. Tropical medicinal plants are rich in polysaccharides, polyphenolics and secondary metabolites that are reported to interfere with the successful RNA isolation. Conventional approaches for the extraction of functional RNA are often time-consuming and need expensive reagents. Moreover, these methods can yield only poor quality and quantity of functional RNA. In our laboratory, we have aimed at establishing a simple and efficient functional RNA extraction procedure. For this, a sodium dodecyl sulfate (SDS) based protocol free of guanidine isothiocyanate was adopted. The total extracted RNA remains in the upper aqueous phase, at acidic conditions leaving DNA and proteins in the lower organic phase. This principle is used in this protocol and the modifications made in the SDS-acid phenol RNA extraction protocol were found to be helpful for extracting RNA from tissues containing large quantities of secondary metabolites. This method can be completed within 3 hours with purity ranges from 1.8-2.0 as confirmed by A 260/280 spectrophotometric readings. The above-described RNA extraction protocol works well with all the tissues examined so far, where standard RNA isolation methods failed. INTRODUCTIONTropical medicinal plants have been largely exploited for therapeutic purposes by present-day researchers. Effective conservation and sustainable utilization of plant biodiversity is indispensable for meeting the present and future requirements of medicinal plants. These medicinal plants are rich sources of alkaloids, polyphenols, polysaccharides, secondary metabolites, terpenoids and these phyto compounds have dragged the attention of contemporary researchers. Production of valuable therapeutic compounds from medicinal plants could be made possible through metabolic engineering and recombinant DNA technology (Titanji et al. 2007). The application of modern molecular biology techniques may help in better understanding and conservation of medicinal plants. These tools can unravel the various bioactive compounds with therapeutic significance at the molecular level. Good quality functional RNA extraction is the most important step in gene expression analysis studies (Ghangal et al. 2009). However, RNA extraction from tissues having higher polyphenols, polysaccharides and secondary metabolite contents became a difficult task. The phenolic compounds readily get oxidised to form quinones. These quinones can bind easily with nucleic acids and act as a barrier for good quality RNA isolation (Wang et al. 2008).Several RNA isolation protocols for medicinal plants has been developed or modified and certain commercial kits were also introduced (John 1992, MacKenzie e...

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“…According to previous reports (Qi et al, 2009;Ghangal et al, 2009;Bilgin et al, 2009), PVP, β-mercaptoethanol, and absolute ethanol are absolutely necessary to eliminate this impurities. Thus, these chemicals were used to modify the method by Qi et al (2009) to test their effects on RNA isolation from sunflower seeds.…”

Section: Discussionmentioning

confidence: 99%

“…We tried to isolate total RNA from their seeds following the handbooks of RNAiso Plus, RNAiso Plus+RNAiso-mate for Plant Tissue and Trizol, or the method reported by Qi et al (2009), but it is difficult to obtain high-quality RNA, perhaps due to the high levels of lipids, starch, polyphenol, polysaccharide, storage proteins, secondary metabolites, and endogenous RNase in seeds (Salzman et al, 1999;Singh et al, 2003;Azevedo et al, 2003;Li and Trick, 2005;Birtic and Kranner, 2006;Bilgin et al, 2009). According to previous reports (Salzman et al, 1999;Qi et al, 2009;Ghangal et al, 2009;Bilgin et al, 2009), soluble polyvinylpyrrolidone (PVP), β-mercaptoethanol and absolute ethanol were used to modify the method reported by Qi et al (2009) for RNA extraction, and high-quality RNA was obtained.…”

Section: Introductionmentioning

confidence: 99%

An optimized preparation method to obtain high-quality RNA from dry sunflower seeds

Yang²

2011

Genet. Mol. Res.

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ABSTRACT.In an attempt to isolate high-quality, intact total RNA from sunflower (Helianthus annuus) seeds for investigation of the molecular mechanisms of mutations, we tested various procedures, using kits, including RNAiso Plus, RNAiso Plus+RNAiso-mate for Plant Tissue, Trizol, and the Qi method, but no high-quality total RNA of high integrity was obtained with any of these methods, probably due to the high content of polyphenols, polysaccharides, and secondary metabolites in mature sunflower seeds. Modifications were made to the Qi method. To avoid polyphenol oxidation, frozen dry seeds free of the seedcase were ground in a mortar with an equal amount of PVP30, and the fine ground powder was transferred to an extraction buffer with 2% PVP30 (w/v), 5% β-mercaptoethanol (v/v) and LiCl (8 M). A sample homogenate was extracted with chloroform prior to acidic phenolchloroform extraction. The total RNA was precipitated with 1/4 volume of NaAc and 2 volumes of absolute ethanol to prevent contamination by polysaccharides. The yield of total RNA was 29.95 μg/100 mg husked dry seeds; the ratios of A260/A230 and A260/A280 were 2.44 and 2.09, respectively. Electrophoretic analysis clearly showed 28S and 161 ©FUNPEC-RP www.funpecrp.com.br Genetics and Molecular Research 10 (1): 160-168 (2011) A method to obtain RNA from sunflower dry seeds 18S ribosomal RNA bands. Using the extracted RNA, a fragment of the actin gene was successfully expressed by RT-PCR. This modified protocol is suitable for isolating high-quality total RNA from sunflower seeds for molecular research.

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A robust plant RNA isolation method suitable for Affymetrix GeneChip analysis and quantitative real-time RT-PCR

Cited by 64 publications

References 16 publications

DWARF AND LOW-TILLERING Acts as a Direct Downstream Target of a GSK3/SHAGGY-Like Kinase to Mediate Brassinosteroid Responses in Rice

DWARF AND LOW-TILLERING Acts as a Direct Downstream Target of a GSK3/SHAGGY-Like Kinase to Mediate Brassinosteroid Responses in Rice

Simple and efficient method for functional RNA extraction from tropical medicinal plants rich in secondary metabolites

An optimized preparation method to obtain high-quality RNA from dry sunflower seeds

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