An improved method for high-quality RNA isolation from needles of adult maritime pine trees

Azevedo, Herlânder; Lino-Neto, Teresa; Tavares, R. M.

doi:10.1007/bf02772582

Cited by 86 publications

(74 citation statements)

References 9 publications

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“…A P. pinaster cDNA library was constructed using the ZAP Express TM Synthesis Kit (Stratagene) and the ZAP Express TM Gigapack III Gold Cloning Kit (Stratagene) (Azevedo et al, 2003). Screening of the cDNA library was carried out according to the supplier's instructions.…”

Section: Cdna Isolation and Northern Blot Analysismentioning

confidence: 99%

“…Cell suspensions were ground to a fine powder in a mortar using liquid nitrogen. Total RNA extraction was performed using a CTAB-based method (Azevedo et al, 2003). Sample normalization and integrity assessment was carried out by formaldehyde gel electrophoresis with ethidium bromide staining.…”

Section: Cdna Isolation and Northern Blot Analysismentioning

confidence: 99%

“…Full-coding cDNA sequences were obtained by screening a maritime pine cDNA library (Azevedo et al, 2003), resulting in the identification of the genes Fe-Sod1 (acc. No.…”

Section: Phylogenetic Analysis Of P Pinaster Fe-sod and Csapxmentioning

confidence: 99%

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Effect of salt on ROS homeostasis, lipid peroxidation and antioxidant mechanisms in Pinus pinaster suspension cells

2009

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Abstract• In the Pinus genus, information on the effectiveness of oxidative defence mechanisms during exposure to salt is lacking. The effect of salt stress imposition on ROS homeostasis was investigated using maritime pine (Pinus pinaster Ait.) suspension cells as a model system.• Cells were maintained in MS-based medium, exposed to salt (50, 100 and 150 mM NaCl) and analysed for biomass production, evidencing a decreasing growth capacity. Use of 100 mM NaCl imposed severe salt stress without affecting cell viability, being chosen for subsequent studies on the ROS homeostasis of salt shock-treated suspension cells.• Increased total ROS levels were evident on the second day of salt exposure, but a superoxide ion transient burst was immediately noticeable. Additionally, lipid peroxide formation seemed to correlate with superoxide ion breakdown. In-gel superoxide dismutase activity evidenced a FeSOD homodimer with strongly increasing activity between hours 12-48 of salt stress imposition. Subsequently, P. pinaster Fe-Sod1 and csApx1 genes were isolated from a cDNA library and expression was shown to increase within 12-24 h.• Results show that severe salt treatment generates oxidative stress in P. pinaster cells despite the induction of antioxidant systems, and suggest a putative involvement of ROS in salt stress signalling. Mots-clés :H 2 O 2 / pin maritime / NaCl / superoxide Résumé -Effet du stress salin sur l'homéostasie des formes réactives d'oxygène, la peroxydation des lipides et les mécanismes antioxydants dans des suspensions cellulaires de Pinus pinaster.• Les informations sur les mécanismes de défense oxydative du pin en réponse à un stress salin sont rares. L'effet d'une exposition au sel sur l'homéostasie des formes réactives d'oxygène (FRO) a été étudié en utilisant une suspension cellulaire de pin maritime (Pinus pinaster Ait.) comme modèle.

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Section: Cdna Isolation and Northern Blot Analysismentioning

confidence: 99%

Section: Cdna Isolation and Northern Blot Analysismentioning

confidence: 99%

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Effect of salt on ROS homeostasis, lipid peroxidation and antioxidant mechanisms in Pinus pinaster suspension cells

2009

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“…Total RNA from adult needles of Pinus pinaster was extracted using a CTAB based method, followed by chloroform extraction [2]. For cDNA library preparation, poli(A)+ RNA was isolated using Oligo (dT) beads (Dynabeads Oligo (dT) 25 , Dynal) and used in ZAP Express™ cDNA synthesis/Gigapack III Gold cloning kits (Stratagene) according to the manufacturer's instructions.…”

Section: Mrna Purification and Cdna Library Constructionmentioning

confidence: 99%

“…Total RNA extraction was performed using a CTAB based method with chloroform extraction [2]. Formaldehyde gel electrophoresis with ethidium bromide staining allowed for sample normalization and integrity assessment.…”

Section: Northern Analysismentioning

confidence: 99%

Salicylic acid up-regulates the expression of chloroplastic Cu, Zn-superoxide dismutase in needles of maritime pine (Pinus pinaster Ait.)

2004

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-Several studies have supported a major role of salicylic acid in modulating plant response against abiotic and biotic stresses, by induction antioxidant capacity. In this work, a full-length cDNA encoding a Cu,Zn -superoxide dismutase was isolated by screening a Pinus pinaster needle cDNA library. The predicted protein of 215 amino acid residues has a molecular mass of 22.1 kDa and exhibits a N-terminal transit peptide, which putatively targets the protein to the chloroplast. Treatment of pine seedlings with salicylic acid resulted in the increase of chloroplastic Cu,Zn superoxide dismutase transcript levels in needles, suggesting a role of this isoform in salicylic acid-mediated H 2 O 2 increase in chloroplasts. Pinus pinaster / acide salicylique / Cu,Zn-SOD chloroplastique

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Isolation of high‐quality RNA from polyphenol‐, polysaccharide‐ and lipid‐rich seeds

Birtić

Kranner

2006

Phytochemical Analysis

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Seed polyphenols, polysaccharides and lipids often interfere with or degrade RNA, restricting its yield and quality. Existing RNA isolation methods specifically alleviate one or two of these challenges, but are usually designed for a single species rather than for a broad biodiversity. Many protocols also do not eliminate chromosomal DNA, which causes false positives in gene expression studies. The method reported here addresses all of the above challenges. The concentration of the phenol blocker polyvinylpyrrolidone in the extraction buffer was optimised for use on a broader range of species. DNase I was added to eliminate chromosomal DNA and the timing of this step was optimised. Lipids were removed by centrifugation. Polysaccharides and proteins, including excess DNase I, were separated from RNA during ethanol precipitation of nucleic acids. In seeds of five plant species from four taxonomically distant families, significant amounts of RNA were isolated in less than half the time typically required by previously reported methods. Colorimetric tests showed that the isolated RNA was free from interfering contaminants. In addition, reverse transcription-PCR confirmed that the isolated RNA was of appropriate quality and integrity for gene expression studies.

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An improved method for high-quality RNA isolation from needles of adult maritime pine trees

Cited by 86 publications

References 9 publications

Effect of salt on ROS homeostasis, lipid peroxidation and antioxidant mechanisms in Pinus pinaster suspension cells

Effect of salt on ROS homeostasis, lipid peroxidation and antioxidant mechanisms in Pinus pinaster suspension cells

Salicylic acid up-regulates the expression of chloroplastic Cu, Zn-superoxide dismutase in needles of maritime pine (Pinus pinaster Ait.)

Isolation of high‐quality RNA from polyphenol‐, polysaccharide‐ and lipid‐rich seeds

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