Isolation of full-length RNA from a thermophilic cyanobacterium.
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.
Luo
X Z
XZ
University of Memphis, TN 38152, USA.
Stevens
S E
SE
D015152:Blotting, Northern; D011232:Chemical Precipitation; D002725:Chloroform; D003080:Cold Temperature; D000458:Cyanobacteria / Q000201:enzymology / Q000235:genetics*; D004587:Electrophoresis, Agar Gel; D004926:Escherichia coli / Q000235:genetics; D006146:Guanidines; D018021:Lithium Chloride; D008623:Mercaptoethanol; D009691:Nucleic Acid Denaturation; D000439:Pentanols; D019800:Phenol; D012329:RNA, Bacterial / Q000302:isolation & purification* / Q000378:metabolism; D012333:RNA, Messenger / Q000302:isolation & purification* / Q000378:metabolism; D012260:Ribonucleases / Q000378:metabolism; D012263:Ribonucleosides; D013861:Thiocyanates; D017968:Vanadium Compounds
D006146:Guanidines; D000439:Pentanols; D012329:RNA, Bacterial; D012333:RNA, Messenger; D012263:Ribonucleosides; D013861:Thiocyanates; D017968:Vanadium Compounds; D019800:Phenol; C054436:guanidine thiocyanate; D008623:Mercaptoethanol; D002725:Chloroform; C029683:isopentyl alcohol; D012260:Ribonucleases; D018021:Lithium Chloride