lithium-chloride and guanidine-thiocyanate

Isolation of full-length mRNA without degradation is critical in the study of in vivo gene regulation and transcription, cDNA synthesis and reverse transcription (RT)-PCR. It is particularly difficult to isolate full-length mRNA from thermophiles, which have higher turnover rates of mRNA degradation. Mastigocladus laminosus is a thermophilic heterocystous cyanobacterium. The assay of M. laminosus cell lysates showed that RNase activity was high and was resistant to the conventional guanidine thiocyanate and 2-mercaptoethanol denaturation methods. The mRNA isolated by several conventional methods was completely degraded. A method was developed to purify full-length mRNA by a combination of fast cooling, vanadyl-ribonucleoside-complex inhibition, phenol-chloroform-isoamyl alcohol extraction, lithium chloride precipitation and the lysing of cells with the French Press. This method produced high-quality, full-length mRNA in high yield. Purified mRNA was suitable for Northern blotting, cDNA synthesis and RT-PCR. This method could be applicable to other thermophiles in which the RNase activity is high and/or is resistant to guanidine thiocyanate.

Topics: Blotting, Northern; Chemical Precipitation; Chloroform; Cold Temperature; Cyanobacteria; Electrophoresis, Agar Gel; Escherichia coli; Guanidines; Lithium Chloride; Mercaptoethanol; Nucleic Acid Denaturation; Pentanols; Phenol; Ribonucleases; Ribonucleosides; RNA, Bacterial; RNA, Messenger; Thiocyanates; Vanadium Compounds

Topics: Animals; Blotting, Northern; Caseins; Chlorides; Chloroform; Female; Guanidines; Lithium; Lithium Chloride; Methods; Phenols; Rabbits; RNA; RNA, Messenger; Thiocyanates; Urea

A rapid procedure for the isolation of intact total cellular RNA from cultured cells is described. This method combines the simultaneous disruption of cells and extraction of nucleic acids in a single step with the use of phenol and a buffer containing 100 mM LiCl. Total cellular RNA can be isolated in approximately 2 hours. The yield and quality of the RNA is comparable to the more widely employed methods requiring extensive preparatory steps such as extraction using guanidinium thiocyanate and subsequent CsCl gradient centrifugation. The RNA isolated using our procedure contains transcripts up to 10 kb in length and is suitable for Northern analysis. This procedure also yields high-molecular-weight DNA, which is a suitable substrate for restriction endonucleases.

Topics: Adrenal Gland Neoplasms; Animals; Blotting, Northern; Blotting, Southern; Centrifugation, Density Gradient; Cesium; Chlorides; Culture Techniques; DNA; DNA Restriction Enzymes; Guanidines; Lithium; Lithium Chloride; Muscles; Phenols; Pheochromocytoma; Rats; RNA; Thiocyanates; Tumor Cells, Cultured