Rapid treatment of whole cells and RNA viruses for analysis of RNA by slot blot hybridization.
To avoid extensive manipulation for the purification of RNA from cells, several methods were evaluated for the direct release of RNA from influenza virus infected cells and supernatants using slot blot hybridization and non-radioactive probes. Treatment with an equal volume of 10 M aqueous guanidine hydrochloride produced the best hybridization signal. Less, but significant amounts of RNA were also released using the following treatments: dilute alkali (final concentration of 0.16 M NaOH) or 100 degrees C/5 min or RNA sample buffer containing formamide/formaldehyde, then heating at 65 degrees C/10 min. Despite the presence of large amounts of cell debris, RNA from guanidine hydrochloride treated whole cell extracts bound quantitatively to the positively charged nylon membranes. The sensitivity of RNA detection when whole cell extracts treated with guanidine hydrochloride were probed with a digoxigenin labelled cDNA probe was similar to the detection of RNA in highly purified, protein free samples. Three positively charged membranes were tested (from Amersham, ICN and Boehringer Mannheim) using two alkaline phosphatase substrates, NBT-X phos, and a chemiluminescent substrate, 3-(2'-spiroadamantane)-4-methoxy-4-(3'-phosphoyloxy)-phenyl- 1-1,2-dioextane (AMPPD) and a peroxidase substrate, tetramethylbenzidine (TMB). The Boehringer Mannheim membrane had the highest sensitivity for the alkaline phosphatase substrates, but the peroxidase reaction with the TMB substrate was the most consistently sensitive, irrespective of which membrane was used. The ability to quantitatively detect RNA from whole cells without any purification will allow the rapid screening of large numbers of samples for specific RNA species in research or diagnostic laboratories.
McKimm-Breschkin
J L
JL
Biomolecular Research Institute, Parkville, Australia.
D000818:Animals; D002460:Cell Line; D004285:Dogs; D009693:Nucleic Acid Hybridization; D012328:RNA Viruses / Q000235:genetics*; D012367:RNA, Viral / Q000032:analysis*
D012367:RNA, Viral