A simple, rapid and effective method for total RNA extraction from Lentinula edodes

Li, Junhui; Tang, Chuanhong; Song, Chunyan; Chen, Mingjie; Feng, Zhen; Yingjie, Pan

doi:10.1007/s10529-006-9074-y

Cited by 23 publications

(17 citation statements)

References 10 publications

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“…3c). The functional intactness of isolated mRNAs can be measured by RT-PCR analysis since reverse transcriptase is highly sensitive to impurities (Li et al 2006;Ghangal et al 2009). As shown in Fig.…”

Section: Resultsmentioning

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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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: Resultsmentioning

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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“…Macroalgae are rich in phenolics, polysaccharides, and other compounds, which hinder the purity of RNA (Sharma et al 2002;Joubert and Fleurence 2005;Li et al 2006;Vanessa et al 2008). In this study, high-qualified E. prolifera RNA was successfully purified by the modified method of CTAB.…”

Section: Discussionmentioning

confidence: 99%

Molecular Cloning and Analysis of a Cytosolic Hsp70 Gene from Enteromorpha prolifera (Ulvophyceae, Chlorophyta)

Yao

et al. 2010

Plant Mol Biol Rep

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Heat shock protein 70 (Hsp70) is one of the important members of heat shock protein (Hsp) families and plays essential roles in folding nascent protein, translocation, refolding denatured protein, protein degradation, adverse stress resistance, and so on. In this study, homologous cloning coupled with the rapid amplification of cDNA ends was used to clone full-length cytosolic heat shock protein 70 of Enteromorpha prolifera (designed as EPHsp70). Bioinformatics was used to analyze structural feature, homologous relationship, and phylogenetic position of EPHsp70. The full length of EPHsp70 cDNA was 2,265 bp, with a 5′ untranslated region of 65 bp, a 3′ untranslated region of 217 bp, and an open-reading frame of 1,983 bp encoding a polypeptide of 660 amino acids with an estimated molecular weight of 71.39 kDa and an estimated isoelectric point of 5.03. EPHsp70 had five degenerate repeats of tetrapeptide GGMP and three typical Hsp70 signature motifs. The C-terminus amino acid sequence of cytosolic EPHsp70 was EEVD, and the conservation of EPHsp70 of N-terminus was higher than that of C-terminus. The homology between EPHsp70 and the cytosolic Hsp70s of other algae and land plants was more than 70%.Keywords Enteromorpha prolifera . Heat shock protein 70 . Molecular cloningUnder inherently stressful environment, intertidal seaweeds have to face different stress factors such as high temperature, low temperature, ultraviolet radiation, and desiccation with the turning tides and alterations caused by diurnal, seasonal, and meteorological variations. In the process of adaptation, seaweeds must make appropriately physiological and biochemical changes in order to deal with environmental stresses, at the same time, seaweeds upregulate expression of stress genes and downregulate expression of protein synthesis-related genes to resist abiotic stresses (Collén et al. 2007). Organisms respond to stress at the cellular level with the rapid synthesis of a number of so-called stress proteins (heat shock proteins (Hsps)). Investigations of stress response in nonmobile organisms such as seaweeds are especially interesting because they are unable to escape from unfavorable conditions. Therefore, it is meaningful to study the role of Hsps in adverse stress resistance mechanism of seaweeds. Stress responses have been studied in few species of seaweeds to date (Vayda and Yuan 1994;Lewis et al. 1998;Ireland et al. 2004;Li and Brawley 2004;Roeder et al. 2005;Collén et al. 2007; Henkel and Hofmann 2008a, b;Fu et al. 2009).Hsps are highly conserved throughout evolution and play an essential role in protecting cells, folding and translocating nascent proteins, refolding denatured proteins, disassembling already formed protein aggregates, and so on under both stress and nonstress conditions (Nelson et al

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“…To the best of our knowledge there are no reports on RNA yields from Trichoderma, thus preventing a direct comparison. (Li et al, 2006). To evaluate the suitability of isolated RNA with the present protocol in downstream molecular biology procedures, total RNA was used for RT-PCR analysis (reverse transcription is highly sensitive to impurities) and the Actin gene was amplified (Fig.…”

Section: Resultsmentioning

confidence: 99%

A rapid and versatile method for the isolation of total RNA from the filamentous fungusTrichoderma sp.

2008

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RNA isolation is essential to study gene expression at the molecular level. However, RNA isolation is especially difficult in organisms such as filamentous fungi which contain compounds that bind and/or co-precipitate with RNA. No commercial kit is yet available for the isolation of high-quality RNA from these organisms. Furthermore, because of the large amounts of polysaccharides, the common protocols for RNA isolation usually result in poor yields when applied to Trichoderma. A simple and fast protocol for the isolation of RNA with high yields that effectively remove contaminating substances is introduced here. The protocol is based on the SDS/phenol cellular sample treatment method, with the following modifications: a phenol/chloroform extraction step for removal of proteins, DNA, and polysaccharides by precipitation without β-mercaptoethanol and liquid nitrogen. This procedure may be useful for the isolation of RNA from other filamentous fungi and may serve as an important tool for the molecular analysis of these organisms.

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A simple, rapid and effective method for total RNA extraction from Lentinula edodes

Cited by 23 publications

References 10 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.)

Molecular Cloning and Analysis of a Cytosolic Hsp70 Gene from Enteromorpha prolifera (Ulvophyceae, Chlorophyta)

A rapid and versatile method for the isolation of total RNA from the filamentous fungusTrichoderma sp.

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