Transcription profiling by array of HeLa cells after miR-31 mimic or miR-31 inhibitor transduction.
ABSTRACT: Expression profiling of HeLa cells transduced stably with pre-miR-31 mimic, miR-31 inhibitor and parental HeLa cells was performed using Human Whole Genome OneArray v6.1 (Phalanx Biotech Group, Taiwan, China). Four hybridizations for each group were performed, with two biological and two technical replicates. Briefly, the signal intensity of each spot was loaded into the Rosetta Resolver System (Rosetta Biosoftware, Cambridge, MA, USA) for data analysis. The technical repeat data were tested by Pearson's correlation coefficient calculation to check the reproducibility (R>0.95). Normalized spot intensities were transformed to gene expression log2 ratios between the control and treatment groups.
Project description:Human lactoferrin (LF) is a multifunctional protein involved in immunomodulation, cell growth, and differentiation. In addition to the secreted form (sLF), an alternatively spliced form (ΔLF) that lacks the signal sequence and downregulated in cancer was found. This study was carried out to identify and compare signaling networks provoked by the two LF isoforms. To do this, the two forms were overexpressed in HEK293 cells using the flp-in/tet-on system and genome-wide expression analysis of 18,367 genes was conducted. Secreted form (sLF) or alternatively spliced form (ΔLF) were overexpressed in HEK293 cells using the flp-in/tet-on system and genome-wide expression analysis of 18,367 genes was conducted.
Project description:Current study will help us to elucidate the differential gene expression profiles of two different subsets of monocytes stimulated with gamma-M. tuberculosis CD14hiCD16- and CD14loCD16+ monocytes were isolated from human peripheral blood using magnetic beads for CD14 and CD16. 1.5 million cells were cultured in 1 ml of complete RPMI medium and stimulated with 10 microgram/ml of gamma-M. tuberculosis for 48 hours. After 48 hours cells were harvested and RNA was isolated from control and stimulated samples from CD14hiCD16- and CD14loCD16+ monocytes. So total we had 4 samples: 1. Control CD14hiCD16- 2. Stimulated CD14hiCD16- 3. Control CD14loCD16+ monocytes and 4. CD14loCD16+ monocytes. RNA from all these above 4 samples were sent to Phalanx Biotech Group for whole genome microarray.
Project description:Microarray analysis of whole lung tissue from three mouse models of asthma Acute asthma was produced by immunization twice, one week apart, of Balb/C mice 8-12 weeks of age with Aspergillus species (5 μg) in adjuvant (1:1 vol/vol). Adjuvant was aluminum and magnesium hydroxide (Pierce). Asthma was initiated by three consecutive intranasal exposures to Aspergillus species (5μg in 15μL saline) and asthma was evaluated 72 hours after the final exposure. Tolerant asthma was produced by intranasal delivery of Aspergillus species (5μg in 15μL saline) twice a week for six consecutive weeks to Balb/C mice 8-12 weeks of age. Asthma was evaluated after mice were rested for an additional three weeks. Chronic asthma was produced by intranasal delivery of the Dustmite/ Ragweed/ Aspergillus (5,50,5ug in 15ul saline) mixture twice a week for six consecutive weeks in female mice Balb/C mice 8-12 weeks of age. Asthma was evaluated 3 weeks after cessation of allergen exposure. Saline control mice were treated intransally with 15ul of saline twice a week for 6 weeks. Asthma was assesed 3 weeks after the final exposure. n=3 samples/ mouse model.
Project description:Transcriptional responses in the gut of the main malaria vector Anopheles gambiae following oral bacterial infection with the entomopathogen Serratia marcescens were identified using DNA microarrays. S. marcescens is a common member of the mosquito gut microbiota, found in both laboratory reared and field collected mosquitoes, that can be potentially pathogenic as in Drosophila (Nehme et al., 2007), while it has been shown to influence the outcome of Plasmodium infections (Bando et al., 2013). S. marcescens belongs to the Enterobacteriaceae family, members of which have been shown to influence malaria transmission dynamics (Cirimotich et al., 2011, Boissiere et al., 2012). To further investigate the interactions between S. marcescens and the mosquito host, likely to shape, directly or indirectly, malaria transmission dynamics, An. gambiae mosquitoes, from the recently established N'gousso M form laboratory colony that retains much of the genetic variation of field mosquitoes, were antibiotic treated for 5 days and subsequently orally infected with the Db11-GFP strain of S. marcescens. Bacteria-fed mosquitoes were selected 2 days post infection, and, 3 days post infection, guts from bacteria-fed mosquitoes were dissected. Uninfected control mosquitoes were treated in the same way. Differential expression in the gut of S. marcescens infected mosquitoes, compared to uninfected controls, was identified by hybridizing labelled complementary RNA, derived from total RNA extracted from the respective gut pools, in customized Agilent 4x44k gene expression microarrays, comprising oligonucleotide probes encompassing all An. gambiae annotated genes of the AgamP3.6 release, with each probe represented in duplicate.
Project description:The present study reports the genetic and biochemical characterization of a dominant glossy mutant allele (BnaA. GL) in B. napus that results in a glossy phenotype. Results from transmission electron microscopy and scanning electron microscopy revealed the GL mutant exhibits reduced deposition of the cuticle layer, which was confirmed by a cuticular wax analysis. The wax compositional analysis revealed an increase in aldehydes but a severe decrease in alkanes, ketones and secondary alcohols. Genetic mapping narrowed the BnaA. GL gene to the end of A9 chromosome, where a gene homologous to ECERIFERUM1 (CER1) in Arabidopsis locates.<br><br>Then, we conducted a microarray analysis to find the differentially expressed genes between normal phenotype and glossy plants. Two comparisons were performed: wild type parent VS. the GL parent, and the bulked normal phenotype DH lines VS. the bulked glossy DH lines. The DH lines are generated from F1 plants of two parents, and RNA samples from three DH lines were combined to make a bulked sample for each phenotype. <br><br>Although no discernible mutation was apparent in the B. napus gene, this cDNA microarray chip assay revealed coordinated down regulation of genes encoding enzymes of the cuticular wax biosynthetic in the glossy mutant with BnCER1 being one of the most severely suppressed genes.
Project description:Cancer stem-like cells are defined as small population of cancer cells which has, higher tumor-initiating ability, self-renewing ability and differentiation ability. In this experiment, transcription profile of cancer stem-like cells derived from human colon cancer line cell SW480 was investigated. Cancer stem-like cells were isolated as side population (SP) cells by Hoechst33342 dye staining from human colon cancer line cell SW480. Non-cancer stem-like cells were isolated as main population (MP) cells. RNAs were isolated from SP cells and MP cells derived from SW480 cells and array experiment was perfromed in dye swaped fashion.
Project description:We used the commercially available amino-allyl RNA amplification Kit ver,2 (High Yield Type) (SIGMA-ALDRICH). Purified total RNA (3 ﾵg) was reverse-transcribed to generate double-stranded cDNA using an oligo dT T7 promoter primer and reverse transcriptase. Next, cRNA was synthesized using T7 RNA polymerase, which simultaneously incorporated Cy3- or Cy5-labeled cytidine triphosphate. During this process, the samples of SP cells were labeled with Cy5, whereas the non-SP cells were labeled with Cy3 as control cells. Quality of the cRNA was again checked using the Nano Drop. Cy3-labeled cRNA and Cy5-labeled cRNA were combined and then fragmented in a hybridization cocktail (SIGMA-ALDRICH). Then the labeled cRNAs were hybridized to a 60-mer probe oligonucleotide microarray and incubated for 20 h ours at 50 ﾰC. The fluorescent intensities were determined by a Genepix 4000B Microarray Scanner (Axon, US).
Project description:Analysis of mRNA changes in HeLa cells following Ago2 or Dicer depletion. Dicer, a cytoplasmic RNase III, generates the mature form of small RNAs including microRNA. Ago2 is a component of an effector complex of microRNA. Keywords: gene expression array-based (RNA / in situ oligonucleotide) siRNA against Ago2 or Dicer were trasnfected into HeLa cells. siRNA against GFP was used as a control. Biologically duplicated total RNAs were prepared from HeLa cells, 24 hrs and 48 hrs after siRNA transfection.