Improved method for the isolation of RNA from bacteria refractory to disruption, including S. aureus producing biofilm.
The development of fast, reliable and inexpensive phenol protocol is described for the isolation of RNA from bacterial biofilm producers. The method was tested on Staphylococcus aureus (S. aureus) and other biofilm-producing gram-negative microorganisms and provided the highest integrity of RNA recovery in comparison to other methods reported here. In parallel experiments, bacterial lysis with Qiagen, NucleoSpin RNAII, InnuREP RNA Mini, Trizol and MasterPure RNA extraction Kits using standard protocols consistently gave low RNA yields with an absence of integrity. The boiling method presented here yielded high concentration of RNA that was free from 16S and 23S rRNA, contained 5S RNA. Higher yields due to improved biofilm bacterial cell lysis were achieved with an added hot phenol incubation step without the need for a bead mill or the enzyme. This method when used in conjunction with the Qiagen RNeasy Mini kit, RNA isolation was a success with greater integrity and contained undegraded 16S and 23S rRNA and did not require further purification. Contaminating DNA was a problem with the RNA processing samples; we used quantitative real-time PCR (RT-qPCR) to measure the recovery of RNA from bacterial biofilm cells using the method described here.
Department of Medical Microbiology and Parasitology, Faculty of Medicine and Health Sciences, Universiti Putra Malaysia, 43400 Serdang, Selangor, Malaysia. salmanatshan@yahoo.com
Salman Sahab
SS
Atshan
Mariana Nor
MN
Shamsudin
Leslie Than Thian
LT
Lung
King Hwa
KH
Ling
Zamberi
Z
Sekawi
Chong Pei
CP
Pei
Ehsanollah
E
Ghaznavi-Rad
D018441:Biofilms; D019800:Phenol; D012329:RNA, Bacterial / Q000302:isolation & purification*; D060888:Real-Time Polymerase Chain Reaction; D013211:Staphylococcus aureus / Q000235:genetics*
D012329:RNA, Bacterial; D019800:Phenol