RNA EXTRACTION FROM STREPTOCOCCUS MUTANS BIOFILMS

Background: RNA extraction with efficient quality and quantity is very important for RT PCR, hybridization on northern blotting and gene expression analysis by real time PCR. Soluble and insoluble extracellular polysaccharides in Streptococcus mutans biofilm cells interfere with RNA extraction procedures. Therefore, finding efficient methods for polysaccharide removal, RNA extraction and purification is necessary for RNA based molecular research. The aim of present study was to evaluate the efficary of a few methods in RNA extraction from Streptococcus mutans.
Methods: In this research we used Streptococcus mutans ATCC 35668 and one clinical isolate of S. mutans from dental plaque. RNA extraction was carried out from biofilms form in 24 well polystyrene microtiter plate using 3 methods. 1: Routine method for RNA extraction from planktonic cells with RNX-Plus solution (Cinagen), 2: Using HYBAID ribolyser Kit, instrument and tubes containing pearls. 3: Using HYBAID ribolyser instrument and RNX-Plus solution. Finally, determination of the isolated RNA purity and integrity was done.
Results: The results showed that RNA extraction from biofilm cells of S. mutans is challenging because of extracellular polysaccharides and the current RNA isolation protocols from planktonic cells (eg. protocol 1) are not suitable for biofilm cells. For this reason, we used HYBAID ribolyser instrument and tubes containing glass and plastic pearls with different size and shapes for maximum cell lysis without disturbing the RNA. The mean photo absorbtion from extracted RNA in latter methods showed statistical significant difference compared to current in-use method (P<0.05).
Conclusion: RNA extraction from S.mutans biofilm cells needs techniques with maximum Polysaccharide removal and maximum cell lysis without disturbing the RNA. The 2^nd and 3^rd methods were more  effective than 1^st one. The 3^rd protocol is recommended due to it’s lower cost.