RNA EXTRACTION FROM SOIL BACTERIUM PSEUDOMONAS PUTIDA AND GREEN ALGA RAPHIDOCELIS SUBCAPITATA AFTER EXPOSURE TO NANOSCALE ZERO VALENT IRON

Soil bacterium Pseudomonas putida and green alga Raphidocelis subcapitata are often used in ecotoxicology studies, including assessment of nanomaterials. Exposure of these microorganisms to reactive nZVI, however, reduce the quality of extracted RNA for further transcriptomic analysis. In this study, we compare RNA extraction using eight extraction protocols. The extraction using the commercial column kits was often unable to elute both the RNA species in a single elution, whereas phenol-chloroform method using RNAzol recovered both small RNA and large RNA from P. putida. On the other hand, the column based RNA extraction kit from ISOLATE II RNA Mini gave the best yield approximately of 1 mu g/mu l of RNA and RIN 9.0. RNA extraction from R. subcapitata was more complicated when we used a similar column based RNA extraction system, it required additional and thorough cell lysis steps that included cell lyophilisation and ribolization prior to phenol-chloroform extraction method. Then the yield of an effective RNA concentration ranged from 300 ng/pl to 980 ng/pl, giving the RIN 5.5-7.8. The final protocols solved most of the extraction problems, and allow experiments involving RNA gene expression analysis of microorganisms exposed to reactive nZVI.