An improved RNA isolation method for succulent plant species rich in polyphenols and polysaccharides

Gehrig, Hans; Winter, Kai-Uwe; Cushman, John C.; Borland, Anne M.; Taybi, Tahar

doi:10.1007/bf02825065

Cited by 144 publications

(106 citation statements)

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“…Phenolic compounds present in the tissue samples readily get oxidised to form quinones (Gehrig et al 2000, DjamiTchatchou & Straker 2011. These quinones can bind easily with nucleic acids and act as a barrier for good quality RNA isolation.…”

Section: Discussionmentioning

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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Section: Discussionmentioning

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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“…Total RNA from seeds or seedlings was prepared using the RNeasy Plant Mini Kit (Qiagen) with 1% polyethylene glycol added to the RLC buffer for seeds as described by Gehrig et al (2000). One microgram of total RNA was treated with DNase I (Invitrogen) and transcribed into complementary DNA (Reverse Transcription System; Promega).…”

Section: Qrt-pcrmentioning

confidence: 99%

Identification of Direct Targets of FUSCA3, a Key Regulator of Arabidopsis Seed Development

2013

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FUSCA3 (FUS3) is a B3 domain transcription factor that is a member of the LEAFY COTYLEDON (LEC) group of genes. The LEC genes encode proteins that also include LEC2, a B3 domain factor related to FUS3, and LEC1, a CCAAT box-binding factor. LEC1, LEC2, and FUS3 are essential for plant embryo development. All three loss-of-function mutants in Arabidopsis (Arabidopsis thaliana) prematurely exit embryogenesis and enter seedling developmental programs. When ectopically expressed, these genes promote embryo programs in seedlings. We report on chromatin immunoprecipitation-tiling array experiments to globally map binding sites for FUS3 that, along with other published work to assess transcriptomes in response to FUS3, allow us to determine direct from indirect targets. Many transcription factors associated with embryogenesis are direct targets of FUS3, as are genes involved in the seed maturation program. FUS3 regulates genes encoding microRNAs that, in turn, control transcripts encoding transcription factors involved in developmental phase changes. Examination of direct targets of FUS3 reveals that FUS3 acts primarily or exclusively as a transcriptional activator. Regulation of microRNA-encoding genes is one mechanism by which FUS3 may repress indirect target genes. FUS3 also directly up-regulates VP1/ABI3-LIKE1 (VAL1), encoding a B3 domain protein that functions as a repressor of transcription. VAL1, along with VAL2 and VAL3, is involved in the transition from embryo to seedling development. Many genes are responsive to FUS3 and to VAL1/VAL2 but with opposite regulatory consequences. The emerging picture is one of complex cross talk and interactions among embryo transcription factors and their target genes.

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“…Total RNA was isolated from leaves collected at midnight (01:00) from the three CAM species, using guanidine hydrochloride or a commercial kit (RNeasy Plant Mini kit, Qiagen, Japan) precipitating with 1-2% (w/v) HMW polyethylene glycol 20,000 (Gehrig et al, 2000). …”

Section: Total Rna Isolation and Race Amplificationmentioning

confidence: 99%

Regulatory Properties of Phosphoenolpyruvate Carboxylase in Crassulacean Acid Metabolism Plants: Diurnal Changes in Phosphorylation State and Regulation of Gene Expression

Theng

Agarie

Nose

2007

Plant Production Science

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: Regulatory properties of phosphoenolpyruvate carboxylase (PEPC, EC 4.1.1.31) in three CAM species, Kalanchoë pinnata, K. daigremontiana and Ananas comosus (pineapple) were examined. PEPC activity in the leaves of the three CAM species exhibited diurnal changes peaking during the first 2-h of darkness in Kalanchoë species and at midnight in pineapple, and then decreasing drastically until dawn. The oscillations of PEPC activity were far greater in Kalanchoë species than in pineapple. In the presence of 2 mM malate, the activity of PEPC decreased in all three CAM species, but the sensitivity of PEPC to malate was markedly different between pineapple and the Kalanchoë species. The malate sensitivity was 2-to 3-times higher in pineapple than in the Kalanchoë species during the dark period, but it was almost the same during the light period. PEPC in the three CAM species was phosphorylated only during the dark period. PEPC proteins were highly phosphorylated during the first 2-h of darkness in Kalanchoë species and at midnight in pineapple, and then they decreased drastically during the latter part of darkness. CAM-specific isoforms of PEPC in the leaves of the three CAM species contained a highly conserved phosphorylation site of Ser-11 at the N-terminus. These PEPC isoforms displayed diurnal changes in transcript abundance, with the peak of transcripts occurring during the dark period. The day/night changes in PEPC transcript abundance were mirrored by changes in the PEPC protein and corresponding enzyme activity over the diurnal cycle. These findings suggest that the diurnal regulation in PEPC activity is determined by the amount of PEPC protein as well as the posttranslational control in these CAM species.

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An improved RNA isolation method for succulent plant species rich in polyphenols and polysaccharides

Cited by 144 publications

References 10 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

Identification of Direct Targets of FUSCA3, a Key Regulator of Arabidopsis Seed Development

Regulatory Properties of Phosphoenolpyruvate Carboxylase in Crassulacean Acid Metabolism Plants: Diurnal Changes in Phosphorylation State and Regulation of Gene Expression

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