A method for RNA isolation from marine macro-algae

Su, Xing; Gibor, Aharon

doi:10.1016/0003-2697(88)90068-1

Cited by 70 publications

(41 citation statements)

References 20 publications

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“…We suspected that inhibition of amplification reactions was at least in part the result of polyphenolics, which inhibit PCR (19). Polyphenolics are found in the leaves, bark, and fruit of most plants and reportedly copurify with nucleic acids during extraction from plant material and sludge (23,37). PVP binds polyphenolics, and adding PVP-40 reduces inhibition of amplification by polyphenolics when included in an RNA extraction (15,36,37).…”

Section: Resultsmentioning

confidence: 99%

Expression of Putative Virulence Factors of Escherichia coli O157:H7 Differs in Bovine and Human Infections

et al. 2006

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Escherichia coli O157:H7 is a commensal organism in cattle, but it is a pathogen in humans. This differential expression of virulence suggests that specific virulence factors are regulated differently in human and bovine hosts. To test this hypothesis, relative real-time reverse transcription-PCR was used to relate the expression of several putative virulence genes (eae, espA, stx 2 , rfbE, ehxA, and iha) to that of the "housekeeping" gene gnd during natural human and experimental bovine infection with E. coli O157:H7. We examined these genes in fecal samples from eight humans and four calves. iha and espA were significantly more expressed in bovine infections. rfbE and ehxA appeared to be more highly expressed in human infections, though these differences did not achieve statistical significance. Our results support the hypothesis that some virulence-associated genes of O157:H7 are differentially expressed in a host-specific manner.Escherichia coli O157:H7, a human pathogen that causes diarrhea, bloody diarrhea, and the hemolytic uremic syndrome (HUS), has acquired multiple virulence factors that might aid its pathogenicity. Principal among these are the phage-encoded Shiga toxins (Stx proteins), which inhibit protein synthesis in eukaryotic cells (41), and the LEE pathogenicity island, which encodes products associated with formation of attaching-and-effacing lesions at the host surface (reviewed in reference 13). E. coli O157:H7 also has a 92-kb nonconjugative plasmid that encodes several putative virulence factors, including enterohemolysin (encoded by ehxA, also termed hlyA [1,5]), a member of the RTX family of exoproteins. E. coli O157:H7 also expresses the IrgA homologue adhesin (Iha), which was recently identified as a virulence factor in uropathogenic E. coli (16,38).Some of these factors can affect animal hosts. For example, E. coli O157:H7 causes attaching-and-effacing lesions on cultured bovine cells (31) and in neonatal calves (8). Despite this, E. coli O157:H7 is not known to cause natural disease in any host except humans. This discrepancy suggests the hypothesis that specific virulence factors of E. coli O157:H7 are regulated differently when the bacteria are in the human rather than the bovine gut. To test this hypothesis, real-time reverse transcription-PCR (RT-PCR) was employed to examine gene expression in fecal samples from children infected with E. coli O157:H7 and from experimentally infected calves. The resulting data support the hypothesis that virulence genes are differentially expressed in the human and bovine hosts.

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Section: Resultsmentioning

confidence: 99%

Expression of Putative Virulence Factors of Escherichia coli O157:H7 Differs in Bovine and Human Infections

et al. 2006

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“…The total RNA from the flowers was extracted as reported [21], with minor modifications. In short: the Pj-inflorescence powders were homogenised by vortexing with glass beads in lysis buffer (2% SDS, 1% 2-mercaptoethanol, 50 mM EDTA, 150 mM T&borate, pH 7.5), 3 ml buffer/g tissue and quickly mixed with 0.25 buffer volume of 100% ethanol and 0.9 buffer volume of 5 M potassium acetate (K+).…”

Section: Total Andpoiy(a)+ Mrna Preparationmentioning

confidence: 99%

“…The total cellular and poly(A)+ RNAs have been prepared as described above [21,22]. RNAs were fractionated on a 1% agarose-formaldehyde gel and transferred to Hybond-N filters as recommended by manufactures.…”

Section: Northern Blot Analysismentioning

confidence: 99%

cDNA cloning, expression and primary structure of Par j I, a major allergen of Parietaria judaica pollen

et al. 1994

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A 659 bp cDNA clone** coding for an allergen of Pj pollen has been isolated from a lambda gt 11 library, and its DNA sequence determined. The cDNA insert showed an open reading frame of 429 bp coding for an allergenic protein of 14,866 Da and a deduced amino acid sequence containing 143 residues. The expressed recombinant protein represented the major allergen Par j I since it reacted with 95% of the sera from Pj-allergic patients (n = 22) and with two Par j I-specific monoclonal antibodies. No similarity with other known DNA and protein sequences has been detected.

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“…b-mercaptoethanol was added to inhibit the RNase and prevent oxidative cross-linking of nucleic acids by polyphenols [40]. Ethanol, sodium acetate, and potassium acetate were used simultaneously to precipitate polysaccharides and remove the lipids [41]. Before the RNA pellets were finally dissolved, 2/3 volume isopropanol and one volume RNAiso were added to aqueous phase and incubated at -20°C overnight to remove the impurities further and selectively precipitate RNA.…”

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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A method for RNA isolation from marine macro-algae

Cited by 70 publications

References 20 publications

Expression of Putative Virulence Factors of Escherichia coli O157:H7 Differs in Bovine and Human Infections

Expression of Putative Virulence Factors of Escherichia coli O157:H7 Differs in Bovine and Human Infections

cDNA cloning, expression and primary structure of Par j I, a major allergen of Parietaria judaica pollen

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

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