strain: PRL2010 growth condition: in vivo condition
Growth protocol
B. bifidum PRL2010 was grown anaerobically in the Man-Rogosa-Sharp (MRS) supplemented with 0.05% (w/v) L-cysteine hydrochloride and incubated at 37°C.
Extracted molecule
total RNA
Extraction protocol
Total RNA was isolated using the macaloid acid method and then treated with DNase. Briefly, cell pellets were resuspended in 1 ml of QUIAZOL and placed in a tube containing 0.8 g of glass beads (diameter, 106 um). The cells were lysed by shaking the mix on a BioSpec homogenizer at 4°C for 2 min (maximum setting). The mixture was then centrifuged at 12,000 rpm for 15 min, and the upper phase containing the RNA-containing sample was recovered. The RNA sample was further purified by phenol extraction and ethanol precipitation according to a previously described method (Sambrook et al., 1989).
Label
Cy5
Label protocol
Five µg of the same pool of total RNA were labeled using RNA Ampulse amplification and labeling kit with Cy5 for Combimatrix arrays (Kreatech Diagnostics, The Netherlands) according to manufacturer instructions.
Hybridization protocol
Prehybridization was performed by incubating the arrays with prehybridization solution (6X SSPE, 0.05% Tween-20, 20mM EDTA, 5x Denhardt's solution, 100 ng/ul Salmon Sperm DNA, 0.05% SDS) for 60 minutes at 45 °C. 5 ug of Labeled aRNA were fragmented by incubation with Fragmentation Solution (40mM Tris Acetate pH 8.1, 100 mM KOAc, 30 mM MgOAc) for 20' at 95°C. Hybridization was performed at 45°C for 16 hours in hybridization solution (6X SSPE, 0.05% Tween-20, 20mM EDTA, 25% DiFormamide, 100 ng/ul Salmon Sperm DNA, 0.04% SDS). Hybridization washings were performed as following: - wash with 6X SSPET wash solution (6X SSPE, 0.05% Tween-20). - wash with 3X SSPET wash solution (3X SSPE, 0.05% Tween-20). - wash with 0.5X SSPET wash solution (0.5X SSPE, 0.05% Tween-20). - wash with PBST was solution (2X PBS, 0.1% Tween-20). - 2 washes with 2X PBS
Scan protocol
Scanning was performed on InnoScan 700 (Innopsys) scanner. Laser was set at 33% power and PMT at 650.
Data processing
Data extraction was carried out using CombiMstrix Microarray Imager software and a quantile normalization of data was performed using Combimatrix Blist v0.6 software. All teh procedures provided were performed as indicated by Combimatrix protocols available at Combimatrix website (www.combimatrix.com).
