An RNA isolation system for plant tissues rich in secondary metabolites

Ghawana, Sanjay; Paul, Asosii; Kumar, Harish; Kumar, Arun; Singh, Harsharan; Bhardwaj, Pardeep Kumar; Rani, Arti; Singh, Ravi S.; Raizada, Jyoti; Singh, Kashmir; Kumar, Sanjay

doi:10.1186/1756-0500-4-85

Cited by 238 publications

(143 citation statements)

References 19 publications

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“…Five popular protocols for RNA extraction viz., Trizol method, Plant RNeasy kit (Qiagen), CTAB method (Chang et al 1993), SDS-Tris saturated phenol method (Ghawana et al 2011) and SDS-acid phenol based method (Hou et al 2011) were tried for isolation of RNA. The optimised protocol is a modification of the SDS-acid phenol protocol (Hou et al 2011).…”

Section: Rna Isolation Methodsmentioning

confidence: 99%

“…RT PCR and qRT-PCR were performed with the total RNA isolated from rhizome tissue following Chang et al 1993;Ghawana et al 2011 and the new protocol only, since the other three protocols either failed or yielded very low concentration of RNA. First strand cDNA was synthesized using total RNA (1 μg) in the presence of Oligo-(dT) 18 primer and Revert Aid Reverse transcriptase (Thermo Scientific) after digesting with 1U of DNase I (Thermo Scientific) as described in supplier's instruction.~950 bp of actin was PCR amplified using forward (5′-GGT GTC ATG GTT GGT ATG GGT C-3′) and reverse (5′-TGC AAT CCA CAT CTGTTG GAA G-3′) primers.…”

Section: Rt-pcr/qrt-pcrmentioning

confidence: 99%

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A simple and efficient protocol for isolation of high quality functional RNA from different tissues of turmeric (Curcuma longa L.)

Deepa

Sheeja

Santhi

et al. 2014

Physiol Mol Biol Plants

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Many experiments in plant molecular biology require processing of a large number of RNA samples and in some cases large quantities are required for a single application. In turmeric, a major spice and medicinal plant, a protocol for RNA isolation is not available. The major difficulty encountered while using other popular protocols is the low yield and quality of RNA which hampers the downstream applications like qRT-PCR, cDNA synthesis and micro RNA isolation. Commercial kits though available are costly and were found to be unsuccessful in case of rhizomes and root tissues that are rich in polyphenols, polysaccharides and alkaloids. It was thus felt that a quick, handy and cheap protocol of total RNA isolation from different tissues of turmeric was required for day to day working in our lab. The new protocol utilizes SDS based extraction buffer including β-mercaptoethanol and PVP with sequential acid phenol:chloroform extraction to remove polyphenols and proteins, followed by the purification with sodium acetate to eliminate polysaccharides. The protocol is simple and can be completed in less than 3 h. The RNA yield from rhizome was higher by more than fivefold with both A 260/280 and A 260/230 ratio in the range of 1.8-2.0. The protocol worked well with leaf, rhizome, pseudostem and root tissues with RIN >7.0 and the isolated RNA could be successfully used for cDNA synthesis, RT-PCR, qRT-PCR and small RNA isolation including microRNA.

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Section: Rna Isolation Methodsmentioning

confidence: 99%

Section: Rt-pcr/qrt-pcrmentioning

confidence: 99%

A simple and efficient protocol for isolation of high quality functional RNA from different tissues of turmeric (Curcuma longa L.)

Deepa

Sheeja

Santhi

et al. 2014

Physiol Mol Biol Plants

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“…The seeds collected were sown in earthen pots, and the germinated saplings maintained under greenhouse conditions at the Indian Institute of Integrative Medicine in Jammu, India (32°449 N longitude, 74°559 E latitude; 305 m asl), were used as source material for the establishment of the in vitro regeneration system and total RNA isolation. Total RNA was isolated (Ghawana et al, 2011) and incubated at 37°C for 30 min with DNase I (Fermentas) to eliminate the traces of genomic DNA. RNA quality was assessed by electrophoresis on 1% formaldehyde agarose gels and by determining the absorbance ratio (A 260/280 ) using a spectrophotometer (AstraAuriga).…”

Section: Plant Selection and Rna Isolationmentioning

confidence: 99%

Functional Promiscuity of Two Divergent Paralogs of Type III Plant Polyketide Synthases

et al. 2016

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Plants effectively defend themselves against biotic and abiotic stresses by synthesizing diverse secondary metabolites, including health-protective flavonoids. These display incredible chemical diversity and ubiquitous occurrence and confer impeccable biological and agricultural applications. Chalcone synthase (CHS), a type III plant polyketide synthase, is critical for flavonoid biosynthesis. It catalyzes acyl-coenzyme A thioesters to synthesize naringenin chalcone through a polyketidic intermediate. The functional divergence among the evolutionarily generated members of a gene family is pivotal in driving the chemical diversity. Against this backdrop, this study was aimed to functionally characterize members of the CHS gene family from Rheum emodi, an endangered and endemic high-altitude medicinal herb of northwestern Himalayas. Two full-length cDNAs (1,179 bp each), ReCHS1 and ReCHS2, encoding unique paralogs were isolated and characterized. Heterologous expression and purification in Escherichia coli, bottom-up proteomic characterization, high-performance liquid chromatography-electrospray ionization-tandem mass spectrometry analysis, and enzyme kinetic studies using five different substrates confirmed their catalytic potential. Phylogenetic analysis revealed the existence of higher synonymous mutations in the intronless divergents of ReCHS. ReCHS2 displayed significant enzymatic efficiency (V max /K m ) with different substrates. There were significant spatial and altitudinal variations in messenger RNA transcript levels of ReCHSs correlating positively with metabolite accumulation. Furthermore, the elicitations in the form of methyl jasmonate, salicylic acid, ultraviolet B light, and wounding, chosen on the basis of identified cis-regulatory promoter elements, presented considerable differences in the transcript profiles of ReCHSs. Taken together, our results demonstrate differential propensities of CHS paralogs in terms of the accumulation of flavonoids and their relative substrate selectivities.Plants, as ground-anchored sessile creatures, invest significant amounts of energy in the production of secondary metabolites to combat environmental pressures. These metabolites often are produced through complex and highly regulated biosynthetic pathways under the influence of different enzymatic machineries. One of the important classes of secondary metabolites is phenylpropanoids. These represent a significant proportion of secondary metabolites, encompassing nearly 20% of total carbon in the terrestrial biosphere. Flavonoids exhibit remarkable chemical diversity and ubiquitous occurrence and play an important role in many aspects of plant development, like flower coloration, photoprotection, pollen development, cell wall growth, and response to stress conditions like UV light protection, herbivory, wounding, interaction with soil microbes, and defense against pathogens (Yu and Jez, 2008;Pandey et al., 2015). Apart from performing numerous imperative roles in plants, flavonoids also have been reported as poten...

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“…However, these protocols were found to be incompatible for RNA isolation from majority of medicinal plant species with higher polyphenol contents. This is because, the secondary metabolites often co-precipitate with RNA and it affects the yield and quality of RNA (Ghawana et al 2011). As RNA molecules are subject to degradation by RNases enzymes (Gasic et al 2004, Hou et al 2011 several RNA isolation protocols need to be tested and modified.…”

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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An RNA isolation system for plant tissues rich in secondary metabolites

Cited by 238 publications

References 19 publications

A simple and efficient protocol for isolation of high quality functional RNA from different tissues of turmeric (Curcuma longa L.)

A simple and efficient protocol for isolation of high quality functional RNA from different tissues of turmeric (Curcuma longa L.)

Functional Promiscuity of Two Divergent Paralogs of Type III Plant Polyketide Synthases

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

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