RNA isolation and microarray analysis were performed as previously described [18 (link)]. Briefly, first, total RNA was isolated by MirVana miRNA Isolation kit (Thermo Fisher Scientific, Waltham, USA). Then a NanoDrop ND-1000 spectrophotometer (NanoDrop Technologies, Wilmington, USA) was subsequently used to perform the quantification of exacted RNA. Total RNA integrity was checked by an Agilent 2100 Bioanalyzer (Agilent, California, USA), Messenger RNA expression microarray was performed on all samples with the Agilent Whole Human Genome Array (Agilent, California, USA). Data acquisition was carried out by the Agilent G2565BA Microarray Scanner System and Agilent Feature Extraction Software (version 9.1). The normalization of probe intensities was performed using GeneSpring GX 11.0. Normalized gene expression value were log-transformed for analysis.
Agilent feature extraction software version 9.1
Manufactured by Agilent Technologies
Agilent Feature Extraction Software (version 9.1) is a software application that automatically processes microarray image data to derive gene expression measurements from Agilent microarray platforms.
Automatically generated - may contain errors
Lab products found in correlation
G2565ba microarray scanner system, by Agilent Technologies (1 mentions)
Agilent whole human genome array, by Agilent Technologies (1 mentions)
Mirvana mirna isolation kit, by Thermo Fisher Scientific (1 mentions)
Nanodrop nd 1000 spectrophotometer, by Thermo Fisher Scientific (1 mentions)
Agilent 2100 bioanalyzer, by Agilent Technologies (1 mentions)
Genespring gx version 12, by Agilent Technologies (1 mentions)
Agilent whole human genome microarray 4 44 k g4112f, by Agilent Technologies (1 mentions)
Trizol reagent, by Thermo Fisher Scientific (1 mentions)
Nd 1000 uv vis spectrophotometer, by Thermo Fisher Scientific (1 mentions)
Rneasy kit, by Qiagen (1 mentions)
2100 bioanalyzer, by Agilent Technologies (1 mentions)
G2505b microarray scanner system, by Agilent Technologies (1 mentions)
Cydye, by GE Healthcare (1 mentions)
4 protocols using agilent feature extraction software version 9.1
1
RNA Isolation and Microarray Analysis
2
Transcriptome Analysis of Paracancerous Tissues
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Total RNA from the paracancerous tissues was isolated with TRIzol reagent (Invitrogen, Carlsbad, CA, USA) and purified using an RNeasy kit (Qiagen, Germantown, MD, USA). RNA was then quantified using an ND‐1000UV‐VIS Spectrophotometer (NanoDrop Technologies, Wilmington, DE, USA), and RNA integrity was determined using a 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA). All RNA samples used in this study showed optical density 260/280 ratios >1.9 and RNA integrity >6. Paracancerous tissue samples were analyzed using an Agilent Whole Human Genome Microarray 4 × 44K (G4112F). All sample labeling, hybridization, washing, and scanning steps were carried out following the manufacturer's specifications. The slides were scanned with an Agilent Microarray Scanner System (G2505B), and the fluorescence intensities were extracted and preprocessed using Agilent Feature Extraction Software version 9.1. Data extraction and annotation were carried out using GeneSpring GX version 12.6.1 (Agilent). The raw data were normalized by the median scale method using the R package “limma” ( www.r-project.org ). Screening probes represented the same gene, and only the probe showing the greatest mean intensity was retained. All raw microarray data and normalized data are publicly available on the GEO website (accession no. GSE101420 ).
3
Comparative Transcriptional Profiling of L. salivarius Mutants
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L. salivarius UCC118 wild-type and both mutant strains were grown in MRS, and RNA was extracted in duplicate, as described above, in exponential and stationary phases. Labelling of cDNA with Cy3 and Cy5 dyes was carried out using a chemical labelling kit (Kreatech), following the manufacturer’s instructions. Microarray slides were hybridized for 16 h at 55 °C and scanned using an Agilent Microarray Scanner system (G2505B) with Agilent scan control software (version 7.0). Agilent feature extraction software (version 9.1) was used to process the image file and the extracted data were further processed using an in-house microarray transform platform, as previously described [2 (link)]. Genes were selected as being significantly changed in expression if their fold change in Cy3/Cy5 ratio was >3 and where the P value was <0.0001. Four microarray conditions were carried out in duplicate: wild-type against ΔlncRNA in exponential phase, wild-type against ΔlncRNA Δ
LSL_1884 in exponential phase, wild-type against ΔlncRNA in stationary phase and wild-type against ΔlncRNA Δ
LSL_1884 in stationary phase.
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L. salivarius UCC118 wild-type and both mutant strains were grown in MRS, and RNA was extracted in duplicate, as described above, in exponential and stationary phases. Labelling of cDNA with Cy3 and Cy5 dyes was carried out using a chemical labelling kit (Kreatech), following the manufacturer’s instructions. Microarray slides were hybridized for 16 h at 55 °C and scanned using an Agilent Microarray Scanner system (G2505B) with Agilent scan control software (version 7.0). Agilent feature extraction software (version 9.1) was used to process the image file and the extracted data were further processed using an in-house microarray transform platform, as previously described [2 (link)]. Genes were selected as being significantly changed in expression if their fold change in Cy3/Cy5 ratio was >3 and where the P value was <0.0001. Four microarray conditions were carried out in duplicate: wild-type against ΔlncRNA in exponential phase, wild-type against ΔlncRNA Δ
LSL_1884 in exponential phase, wild-type against ΔlncRNA in stationary phase and wild-type against ΔlncRNA Δ
LSL_1884 in stationary phase.
4
Porcine Intestinal Microbial Profiling Using PITChip
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Three replicates of each location in each treatment were used for the PITChip analysis. A total of 36 samples from the four treatments and three gut locations were used for analysis. The PITChip is a phylogenetic microarray with >2900 oligonucleotides based on 16S rRNA gene sequences of 627 porcine intestinal microbial species-level phylotypes (P érez Guti érrez 2010). The protocols for amplification, labelling, hybridisation and analysis of the generated data were performed essentially as previously described for the Human Intestinal Tract Chip (Rajilic-Stojanovic et al. 2009) . In brief, the bacterial 16S rRNA genes were amplified using the primers T7prom-Bact-27-for and Uni-1492-rev, the PCR products were transcribed into RNA and the purified resultant RNA was coupled with CyDye (GE Healthcare Life Sciences) before fragmentation and hybridisation to the array. Microarray images were processed using Agilent Feature Extraction Software, version 9.1 (Agilent Technologies). Data normalisation and processing were performed as described previously (Rajilic-Stojanovic et al. 2009) .
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