host: Bos indicus bacteria: Cultured and uncultured bacteria from B. indicus rumen agent: control (no essential oils) incubation: Rumen mixed culture was incubated in vitro for 24 h
Extracted molecule
genomic DNA
Extraction protocol
Metagenomic DNA was extracted from 0.5 ml of each in vitro rumen mixed culture sample using the repeated bead beating (on a Mini-Beadbeater™, BioSpec Products, Bartlesville, OK, USA) and column purification (using QIAamp DNA Stool Mini Kit, Qiagen, Valencia, CA, USA) method. nearly full-length 16S rRNA genes were amplified from each metagenomic DNA sample using the universal primer set 27F (5’-AGA GTT TGA TCM TGG CTC AG-3’) and T7/1492R (5’-TCT AAT ACG ACT CAC TAT AGG GGG YTA CCT TGT TAC GAC TT-3’). The amplicons were purified using a PCR purification Kit (Qiagen, Valencia, CA, USA) and then used in preparation of complementary RNA (cRNA) using a MEGAScript T7 in vitro transcription kit (Ambion, Austin, TX, USA). The cRNA was purified using a MEGAclear kit (Ambion, Austin, TX, USA).
Label
Cy5
Label protocol
The cRNA was labeled with Cy5 at 37oC for 1 hour using a Label IT®uArrayCy3/Cy5 Labeling kit (Mirus, Madison, WI, USA). The labeled cRNA was again purified to remove the free Cy5 dye using a MEGAclear kit.
Hybridization protocol
Microarray hybridization was performed using Agilent Technologies’ Hybridization gasket slides.The hybridization solution and 150 ng labeled cRNA was incubated at 65oC for 5 min and then placed on ice for 5 min. The assembled cassette was placed in a HB-1000 hybridization oven (UVP, LLC) preset at 45oC to allow hybridization for 18 hours with rotation set at 10 rpm.
Scan protocol
Scanned on a GenePix 4000B Scanner (Axon Instruments, Union City, CA). The Cy5 fluorescence signal at each probe spot was measured using the GenePix®Pro 6.0 program (Axon Instruments).
Description
Sample name: CON-rep3 Metagenomic DNA was extracted, amplified using the universal primer set 27F and T7/1492R. The amplicons were purified and used in preparation of complementary RNA (cRNA). The cRNA was purified, and labeledwith Cy5.
Data processing
The local median background signal intensity was subtracted from the median hybridization signal intensity of each separate probe spot. After background subtraction, normalization was performed based on the signal intensity of internal control probes targeting the bovine mitochondrial rRNA gene region.
Essential oils affect populations of some rumen bacteria as revealed by microarray analysis
Data table header descriptions
ID_REF
VALUE
Relative abundance of each operational taxonomic unit was calculated as its probe signal intensity percentage of total bacterial probe signal intensity after background subtraction and normalization.
