An efficient method for isolation of RNA and DNA from plants containing polyphenolics

John, Maliyakal E.

doi:10.1093/nar/20.9.2381

Cited by 240 publications

(117 citation statements)

References 2 publications

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“…Several RNA isolation protocols for medicinal plants has been developed or modified and certain commercial kits were also introduced (John 1992, MacKenzie et al 1997, Sabir 2012. However, these protocols were found to be incompatible for RNA isolation from majority of medicinal plant species with higher polyphenol contents.…”

Section: Introductionmentioning

confidence: 99%

“…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 et al 1997, Sabir 2012. However, these protocols were found to be incompatible for RNA isolation from majority of medicinal plant species with higher…”

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confidence: 99%

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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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Section: Introductionmentioning

confidence: 99%

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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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“…Phenolic compounds are powerful oxidizing agents and bind covalently to the extracted DNA, making it useless for most of molecular manipulations (Porebski et al, 1997;Padmalatha and Prasad, 2006). A high concentration (0.025 g/ml) of PVP mixed in the extraction buffer (Fang et al, 1992;John, 1992;Moller et al, 1992;Lodhi et al, 1995) binds to phenolic compounds and helps in their removal. The superfluous quantities of cellular proteins were managed by triple extended treatment with chloroform-isoamyl alcohol.…”

Section: Discussionmentioning

confidence: 99%

Extraction of DNA suitable for PCR applications from mature leaves of Mangifera indica L.

Azmat

Khan²,

Cheema³

et al. 2012

J. Zhejiang Univ. Sci. B

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Good quality deoxyribonucleic acid (DNA) is the pre-requisite for its downstream applications. The presence of high concentrations of polysaccharides, polyphenols, proteins, and other secondary metabolites in mango leaves poses problem in getting good quality DNA fit for polymerase chain reaction (PCR) applications. The problem is exacerbated when DNA is extracted from mature mango leaves. A reliable and modified protocol based on the cetyltrimethylammonium bromide (CTAB) method for DNA extraction from mature mango leaves is described here. High concentrations of inert salt were used to remove polysaccharides; Polyvinylpyrrolidone (PVP) and β-mercaptoethanol were employed to manage phenolic compounds. Extended chloroform-isoamyl alcohol treatment followed by RNase treatment yielded 950-1050 µg of good quality DNA, free of protein and RNA. The problems of DNA degradation, contamination, and low yield due to irreversible binding of phenolic compounds and coprecipitation of polysaccharides with DNA were avoided by this method. The DNA isolated by the modified method showed good PCR amplification using simple sequence repeat (SSR) primers. This modified protocol can also be used to extract DNA from other woody plants having similar problems.

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“…For particularly challenging plants with high phenolic content, addition of polyvinylpyrrolidone was traditionally used to remove the compounds that bind to DNA following cell lysis 2,5 . As evident here, these complicated, time-intensive steps can be avoided through use of the Phire Plant Direct PCR Kit to detect the target DNA.…”

Section: Discussionmentioning

confidence: 99%

“…In these cases, an additional step to remove the compounds is traditionally required 2,5 . Here, this problem is overcome by using a quick and easy dilution protocol followed by Direct PCR amplification (Figure 1).…”

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confidence: 99%

Genotyping of Plant and Animal Samples without Prior DNA Purification

Chum

Haimes

André

et al. 2012

JoVE

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The Direct PCR approach facilitates PCR amplification directly from small amounts of unpurified samples, and is demonstrated here for several plant and animal tissues (Figure 1). Direct PCR is based on specially engineered Thermo Scientific Phusion and Phire DNA Polymerases, which include a double-stranded DNA binding domain that gives them unique properties such as high tolerance of inhibitors.PCR-based target DNA detection has numerous applications in plant research, including plant genotype analysis and verification of transgenes. PCR from plant tissues traditionally involves an initial DNA isolation step, which may require expensive or toxic reagents. The process is time consuming and increases the risk of cross contamination 1,2 . Conversely, by using Thermo Scientific Phire Plant Direct PCR Kit the target DNA can be easily detected, without prior DNA extraction. In the model demonstrated here, an example of derived cleaved amplified polymorphic sequence analysis (dCAPS) 3,4 is performed directly from Arabidopsis plant leaves. dCAPS genotyping assays can be used to identify single nucleotide polymorphisms (SNPs) by SNP allele-specific restriction endonuclease digestion 3

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An efficient method for isolation of RNA and DNA from plants containing polyphenolics

Cited by 240 publications

References 2 publications

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

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

Extraction of DNA suitable for PCR applications from mature leaves of Mangifera indica L.

Genotyping of Plant and Animal Samples without Prior DNA Purification

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