Rapid and Effective Method of RNA Isolation from Green Microalga Ankistrodesmus convolutus

Thanh, Tran; Omar, Hishamuddin; Abdullah, Md. Pauzi; Quynh, Vu Thi; Noroozi, Mostafa; Kỳ, Huỳnh; Napis, Suhaimi

doi:10.1007/s12033-009-9182-8

Cited by 21 publications

(20 citation statements)

References 14 publications

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“…Currently, the method of isolating RNA from such plant material are: soluble PVP-ethanol precipitation, modified guanidine thiocyanate -phenol -chloroform method [12], improved hot borate method [13], a high molecular polyethylene glycol method, LiCl-2-butoxyethanol -LiCl method, potassium acetate, ethanol insoluble PVP-treatment method, LiCl precipitation method [14], silicon dioxide (silica) -method [15], isothiocyanate -vinegar acid method [16,17], CTAB method [18], Urea-LiCl-PVP method [19] and Urea-ethanol / isopropanol -acetate precipitation method [20] and so on. However, these methods are by phenol -removal of proteins, polysaccharides, polyphenols and secondary metabolites chloroform extraction, and some further added β-ME strong irritant drugs [16,18,20,21], precipitation RNA vinegar salt further remove contaminating polysaccharides [16,21], complicated operation, time-consuming, toxicity, there are some drawbacks.…”

Section: Electrophoresis Test Results and Analysismentioning

confidence: 99%

An Optimized Preparation Method to Obtain High-quality RNA from the Buds of Sunflowers

Feng¹,

Ma²,

Yang³

2016

Proceedings of the 2016 5th International Conference on Environment, Materials, Chemistry and Power Electronics

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Abstract.In an attempt to isolate high-quality, total RNA from flower buds of sunflowers (Helianthus annuus). We have already improved the SDS method. After adding liquid nitrogen, we have to grind the material down. After that we add protein -DNA precipitation, wash it with the addition of different kinds of alcohol, such as 75% alcohol and precipitation. Then we add DEPC water and guanidine hydrochloride blend, LiCl overnight precipitation,and then wash it off with 75% alcohol, finally make it dissolved in 15 µL DEPC water which collocated in minus 80 environment for storage. The total RNA is precipitated with 1/10 volume of NaAc and 2 volumes of absolute ethanol to prevent contamination by polysaccharides. These experimental results show that the total RNA extracted by this method is of high purity and integrity, and the electrophoretic bands are clear. D260/D230, D260/D280 and RNA (g/100mg) are 2.05, 2.11 and 9.52, respectively. Total RNA can amplify the target gene fragment by PT-PCR. This improved method can be used to extract the high-quality RNA from the sunflower buds of the space mutation, in order to study the molecular mechanism of the next step.

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Section: Electrophoresis Test Results and Analysismentioning

confidence: 99%

An Optimized Preparation Method to Obtain High-quality RNA from the Buds of Sunflowers

Feng¹,

Ma²,

Yang³

2016

Proceedings of the 2016 5th International Conference on Environment, Materials, Chemistry and Power Electronics

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“…In fact, CTAB-based methods have recently been used for nucleic acid isolation from polysaccharide-rich plants, and in particular, these methods have been used for RNA isolation (Thanh et al, 2009;Abbasi et al, 2010;Wang et al, 2012). Both A 260 /A 230 and A 260 /A 280 ratios were more than 2 suggesting that the RNA was of high purity and free of polysaccharide/polyphenol and protein contamination, respectively (Table 1) (Wang et al, 2005;Yang et al, 2011 Table 1.…”

Section: Resultsmentioning

confidence: 99%

Short Communication Efficient isolation of high-quality RNA from lotus Nelumbo nucifera ssp nucifera tissues

Zhang¹,

Hao²,

Liang³

et al. 2013

Genet. Mol. Res.

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ABSTRACT. Nelumbo nucifera is widely used as food, as an ornamental, in medicine, and as packing material; it is also reported to have anti-HIV effects and antioxidant capacity. We sought an improved method for extracting high-quality total RNA from different tissues of N. nucifera. Four methods for RNA extraction were assessed for their ability to recover high-quality RNA applicable for evaluation of polyphenol oxidase (PPO) gene expression profiles. The recovery and quality of the RNA obtained from five different tissues by the best CTAB-LiCl method were evaluated through UV light absorbance. Both A 260 /A 280 and A 260 /A 230 absorbance ratios were more than 2.0; the yield ranged from 59.87 to 163.75 μg/g fresh weight. The brightness of the 28S band was approximately twice that of 18S; the latter was also considered as high-quality RNA. The PPO gene fragment (606 bp) was successfully amplified by RT-PCR, demonstrating the integrity of the isolated RNA. The relative expression levels of the PPO gene based on RT-PCR in five tissues of lotus were: rhizome buds (2.66), young leaves (2.42), fresh cut rhizome (2.02), petals (1.80), and petiole (1.65), using housekeeping gene β-actin as an internal control. We concluded that the total RNA isolated by this protocol is of sufficient quality for molecular applications.

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“…There are a large number of successful RNA isolation protocols for different species and different tissues from the same species [15,[28][29][30][31]. However, most of the researches about RNA extraction focus on plants, because the cell walls of plants are very thick and there are more RNA inhibitors such as polyphenols and polysaccharides in [11,12,14,15,19].…”

Section: Discussionmentioning

confidence: 99%

High-Quality RNA Preparation from Rhodosporidium toruloides and cDNA Library Construction Therewith

et al. 2010

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Oleaginous yeast Rhodosporidium toruloides is an excellent microbial lipid producer. Therefore, it is important to develop molecular biology tools to understand the basic mechanism for lipid accumulation and further manipulate the microorganism. High-quality RNA extraction from R. toruloides is particularly challenging due to high level of polysaccharides, lipids, and other secondary metabolites. To obtain an optimal protocol for RNA extraction from R. toruloides, four methods were evaluated. Large difference in RNA yield and quality among these protocols was found. The optimum method was modified RNAiso procedure, where RNA was isolated using liquid nitrogen-RNAiso method with salt precipitation and the addition of b-mercaptoethanol. This method consistently recovered RNA in good quality with high yield. Around 297 lg total RNA per gram of cells was obtained with an average purity measured as A 260 /A 280 of 2.09. A titer of 10 5 cfu/ml could be harvested to construct a full-length cDNA library with the RNA sample in this quality. Electrophoresis gel analysis indicated the fragments ranged from 200 bp to 4.0 kb, with the average size of 1000 bp. Randomly picked clones showed the recombination efficiency at 80%. These results showed that RNA of R. toruloides was successfully extracted for the first time using the modified RNAiso method, and the cDNA library was appropriate for screening the genes related to lipid accumulation.

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Rapid and Effective Method of RNA Isolation from Green Microalga Ankistrodesmus convolutus

Cited by 21 publications

References 14 publications

An Optimized Preparation Method to Obtain High-quality RNA from the Buds of Sunflowers

An Optimized Preparation Method to Obtain High-quality RNA from the Buds of Sunflowers

Short Communication Efficient isolation of high-quality RNA from lotus Nelumbo nucifera ssp nucifera tissues

High-Quality RNA Preparation from Rhodosporidium toruloides and cDNA Library Construction Therewith

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