Two-step IMS was able to enrich E. coli to around 95% from biofilms containing only 8.1% E. coli (2.3 × 106 CFU/ml E. coli and 2.6 × 107 CFU/ml S. maltophilia) (Figure 2B). The results demonstrated the feasibility of using IMS to separate E. coli cells from biofilms. It is important to obtain target cells in high purity from mixed species communities for subsequent cDNA microarray analysis in order to effectively limit cross hybridization. The results showed that a high purity of E. coli cells could be obtained by IMS from different mixed-species communities (suspensions or biofilms) with various amounts
of E. coli cells (0.7-71.3%). Preservation find more of RNA integrity during cell separation Preserving RNA integrity during IMS is critical when collected cells are used for subsequent cDNA microarray analysis. RNAlater (Ambion, Austin, TX) has been used widely to preserve RNA in bacterial cells, but the impact of RNAlater on IMS performance was unknown. The recovery rate of E. coli dropped to 1% if cells remained
in RNAlater during the complete IMS procedure. This may be the result of antibody denaturing by the global protein denaturing reagents present in RNAlater. Alternative products, such as RNAprotect (Qiagen, Germantown, MD), contain similar denaturing reagents and are expected to show similarly reduced recoveries. In order to overcome this problem, RNAlater was removed during SP600125 supplier some steps of the IMS procedure. Samples were stored in RNAlater at 4°C overnight to allow the reagent to penetrate into bacterial cells and to stabilize intracellular RNA. RNAlater was then removed and bacterial cells were resuspended in separation buffer just before incubation with antibody
and microbeads. One-step IMS enriched E. coli to a similar level as shown in Figure 2A and removed over 99% of S. maltophilia cells (data not shown). The results confirmed that the modified protocol did not affect the recovery and purity of E. coli processed by IMS. Pre-stabilization in RNAlater, quick sample processing (~30 min), low working temperature (4°C), and maintaining an RNAase-free environment were combined Protein kinase N1 to limit RNA degradation during IMS, since RNAlater had to be removed during some steps of the IMS procedure. The effectiveness of these strategies in preserving the integrity of RNA was confirmed by observing, using agarose gel electrophoresis, high quality RNA Berzosertib molecular weight extracted from cells treated with the IMS procedure (data not shown). Impact of cell separation on E. coli transcription profiles To evaluate whether gene expression profiles were changed during sample processing (biofilm dispersion) and IMS cell sorting, cDNA microarray analysis was used to compare gene expressions of E. coli cells without dispersion and IMS (unsorted cells) and with dispersion and IMS (sorted cells). To eliminate the possible impact of any non-target RNA (from the small amount (< 5%) of S.