ABSTRACT
The RNA-based study provides an excellent indication of an organism's gene expression profile. Obtaining high-yield and high-purity RNA from Gram-positive and acid-fast bacteria is difficult without high-end kits and facilities. We optimised effective and simple protocol for RNA isolation that is a combination of enzymatic, physical and chemical treatment to disrupt cells. We successfully isolated high quality intact total RNA with yields ranging from 23.13 ± 0.40 to 61.51 ± 0.27 µg and the 260/280 purity ratio of 1.95 ± 0.01 to 2.05 ± 0.01 from Staphylococcus aureus, Staphylococcus epidermidis, Enterococcus faecalis, and Mycobacterium smegmatis. These results represents a significantly enhanced yield and purity compared to other combination of techniques which we performed. Compared to previous studies the yield obtained by this method is high for the studied organisms. Furthermore the yielded RNA was successfully used for downstream applications such as quantitative real time PCR. The described method can be easily optimised and used for various bacteria.
Subject(s)
RNA, Bacterial , RNA, Bacterial/genetics , RNA, Bacterial/isolation & purification , Staphylococcus aureus/genetics , Staphylococcus aureus/isolation & purification , Gram-Positive Bacteria/genetics , Gram-Positive Bacteria/isolation & purification , Staphylococcus epidermidis/genetics , Staphylococcus epidermidis/isolation & purification , Enterococcus faecalis/genetics , Enterococcus faecalis/isolation & purification , Mycobacterium smegmatis/geneticsABSTRACT
BACKGROUND: To identify low molecular weight urinary proteins capable of detecting diabetic nephropathy patients which may predict renal alterations at early stages and prevent it from worsening further. METHOD: Three hundred ninety (390) age-matched subjects were divided into 8 groups depending upon duration of diabetes and the severity of renal damage. Urinary proteome profile of all subjects was determined with the help of microfluidic array. Participants with similar profile were further selected to study proteome map of urinary low molecular weight proteins with the help of 2 dimensional gel electrophoresis. RESULTS: Out of 390 total patients 268 patients showed a similar one dimensional proteomic pattern. Further, two-dimensional urinary proteomic pattern of these patients with molecular weight < 50 kDa was studied. Eight proteins with molecular weight 11, 15, 17, 23, 34, 38 and 46 kDa were identified with MALDI-QTOF. These low molecular weight proteins showed gradual increase in urinary excretion along with the duration of diabetes and severity of renal damage. CONCLUSION: The study concludes that proteomic analysis might be a useful tool for detecting some novel markers capable of detecting patients susceptible to diabetic nephropathy in the early phase.
