Efficient isolation of total RNA from Clostridium without DNA contamination.
Several molecular techniques require high-quality RNA, free from DNA. Various methods have been described to obtain RNA to be used in expression studies or as starting material in differential display-reverse transcriptase (dd-RT-PCR), for which high-quality RNA free from DNA is an essential requirement. In this report, we compare three different methods to isolate RNA from Gram-positive bacteria: (1) An acid-phenol extraction protocol. (2) The "RNeasy mini kit" from QIAGEN (Valencia, CA, USA). (3) The "SV Total RNA Isolation System" from Promega (Madison, WI, USA).The QIAGEN-kit delivers the highest amount of RNA with the highest purity. Slot blot analysis and dd-RT-PCR confirm the absence of DNA contamination and Northern blot analysis and dd-RT-PCR show high quality of the extracted RNA. This RNA extraction method thus addresses current problems by permitting rapid and safe isolation with high yields of intact RNA for subsequent analysis.
Nuyts
S
S
Laboratory of Bacteriology, Rega Institute, K.U. Leuven, Minderbroedersstraat 10, 3000 Leuven, Belgium. Sandra.Nuyts@rega.kuleuven.ac.be
Van Mellaert
L
L
Lambin
P
P
Anné
J
J
D015152:Blotting, Northern; D003013:Clostridium / Q000737:chemistry / Q000235:genetics*; D004269:DNA, Bacterial / Q000737:chemistry / Q000302:isolation & purification; D020869:Gene Expression Profiling; D012329:RNA, Bacterial / Q000737:chemistry / Q000302:isolation & purification*; D020133:Reverse Transcriptase Polymerase Chain Reaction
D004269:DNA, Bacterial; D012329:RNA, Bacterial