sodium-dodecyl-sulfate and guanidine-thiocyanate

Isolation of total RNA from plant materials has been difficult, due to the presence of complex organic substances and the associated pigmentation. In fact, there is a dearth of standardized protocols for isolating total RNA from pollens. To find a simple and reliable method for isolating total RNA from pollen, four methods, viz. phenol/SDS (PS), guanidine HCl (GH), tri-reagent (TR), and modified SDS-betaME (SB) were tested with fresh pollen of Ricinus communis (procured at -70 degrees C) and pollen dried at 30-37 degrees C. The quality and quantity of RNA was superior for the material processed at -70 degrees C. SB gave the highest RNA yield (2.35 mg/g, OD260/280 >2.0), compared to other methods. The results obtained by the SB method were found to be comparable with the widely used tri-reagent method. This was validated with other pollens of Imperata cylindrica and Xanthium strumarium. The yield obtained from graded amounts of pollen was consistent with SB, compared to the TR method. The RNA isolated by SB gave good quality mRNA for synthesizing cDNA. The SDS-betaME method is simple, efficient, and uses less expensive reagents. Hence, we recommend the modified SDS-betaME method for isolating total RNA from pollens.

Topics: Guanidine; Guanidines; Mercaptoethanol; Phenols; Pollen; RNA, Plant; Sodium Dodecyl Sulfate; Thiocyanates

PrPSc, an abnormal conformational isoform of the normal prion protein, PrPC, is the only known component of the prion, a proteinacious agent that causes fatal neurodegenerative disorders in humans and other animals. The hallmark properties of PrPSc are its insolubility in nondenaturing detergents and its resistance to digestion by proteases. Anions such as Congo red (CR) have been shown to reduce the accumulation of PrPSc in a neuroblastoma cell line permanently infected with prions as well as to delay disease onset in rodents when administrated prophylactically. The mechanism by which such anti-prion agents operate is unknown. We show here that in vitro incubation with CR renders native PrPSc resistant to denaturation by boiling SDS. This resulted from PrPSc conformation, since neither the properties of PrPC nor those of predenatured PrPSc were changed by the addition of CR. CR-PrPSc could only be denatured by the addition of acidic 3 M guanidine thiocyanate. Since in vitro conversion experiments have suggested that partial denaturation may be required for PrPSc to serve as template in the PrPC --> PrPSc conversion, we propose that CR inhibits prion propagation by overstabilizing the conformation of PrPSc molecules.

Topics: Animals; Brain; Congo Red; Cricetinae; Guanidines; Mesocricetus; Neuroblastoma; Prions; Protein Denaturation; Scrapie; Sodium Dodecyl Sulfate; Thiocyanates; Tumor Cells, Cultured

Topics: Carbon Dioxide; Drug Contamination; Flavonoids; Gene Expression Regulation, Plant; Guanidines; Indicators and Reagents; Molecular Biology; Phenol; Phenols; Plants; Polymers; Polyphenols; Polysaccharides; RNA, Plant; Sodium Dodecyl Sulfate; Solutions; Spectrophotometry, Ultraviolet; Thiocyanates

Topics: Bacterial Proteins; DNA, Bacterial; Electrophoresis, Agar Gel; Escherichia coli; Guanidines; Indicators and Reagents; Molecular Biology; Muramidase; Polyethylene Glycols; Ribonucleases; RNA, Bacterial; Sodium Dodecyl Sulfate; Thiocyanates