Exon-array profiling of Heat-shock stress response in HeLa cell line
ABSTRACT: The heat-shock stress response was studied at the level of exons using Affymetrix Exon-array profiling for both sense and anti-sense transcripts. Sense transcript profiling was done as per the protocol of Affymetrix Exon 1.0 ST array and anti-sense transcript array profiling was done using a modified protocol (Xijin Ge et al., BMC Genomics. 2008 Jan 22;9:27). In short, for profiling antisense transcripts, first cycle cDNA synthesis and IVT step is skipped. This modified protocol starts directly from the second cycle cDNA synthesis step. The labelled target DNA fragments are in reverse orientation of original mRNAs. Thus the hybridization signals will represent transcripts from the same exonic regions but from the opposite DNA strand. Study was undertaken with 2 biological replicates each for - a) Heat-shock treated Sense b) Heat-shock treated Anti-sense c) Untreated control Sense d) Untreated control Anti-Sense
Project description:The heat-shock stress response was studied at the level of exons using Affymetrix Exon-array profiling for both sense and anti-sense transcripts. Sense transcript profiling was done as per the protocol of Affymetrix Exon 1.0 ST array and anti-sense transcript array profiling was done using a modified protocol (Xijin Ge et al., BMC Genomics. 2008 Jan 22;9:27). In short, for profiling antisense transcripts, first cycle cDNA synthesis and IVT step is skipped. This modified protocol starts directly from the second cycle cDNA synthesis step. The labelled target DNA fragments are in reverse orientation of original mRNAs. Thus the hybridization signals will represent transcripts from the same exonic regions but from the opposite DNA strand. Overall design: Study was undertaken with 2 biological replicates each for - a) Heat-shock treated Sense b) Heat-shock treated Anti-sense c) Untreated control Sense d) Untreated control Anti-Sense
Project description:The repertoire of transcripts that are differentially regulated in response to Heat-shock were studied using Illumina WG-6 v2.0 BeadChip. Study was undertaken with 3 biological replicates for each condition of Heat-Shock Treated and Untreated condition.
Project description:Background: Recent studies have demonstrated that antisense transcription is pervasive in budding yeasts and is conserved between Saccharomyces cerevisiae and S. paradoxus. While studies have examined antisense transcripts of S. cerevisiae for inverse transcription in stationary phase and stress conditions, there is a lack of comprehensive analysis of the conditional specific evolutionary characteristics of antisense transcription between yeasts. Here we attempt to decipher the evolutionary relationship of antisense transcription of S. cerevisiae and S. paradoxus cultured in mid log, early stationary phase, and heat shock conditions. Results: Massively parallel sequencing of sequence strand-specific cDNA library was performed from RNA isolated from S. cerevisiae and S. paradoxus cells at mid log, stationary phase and heat shock conditions. We performed this analysis using a stringent set of sense ORF transcripts and non-coding antisense transcripts that were expressed in all the three conditions, as well as in both species. We found the divergence of the condition specific anti-sense transcription levels is higher than that in condition specific sense transcription levels, suggesting that antisense transcription played a potential role in adapting to different conditions. Furthermore, 43% of sense-antisense pairs demonstrated inverse transcription in either stationary phase or heat shock conditions relative to the mid log conditions. In addition, a large part of sense-antisense pairs (67%), which demonstrated inverse transcription, were highly conserved between the two species. Our results were also concordant with known functional analyses from previous studies and with the evidence from mechanistic experiments of role of individual genes. Conclusions: This study provides a comprehensive picture of the role of antisense transcription mediating sense transcription in different conditions across yeast species. We can conclude from our findings that antisense regulation could act like an on-off switch on sense regulation in different conditions. Transcriptomes of two yeast species under mid-log phase, early stationary phase, and after heat shock treatment were generated by Illumina HiSeq 2000 paired-end sequencing
Project description:aThese experiments address the effects of depleting Hsp90 upon the transcriptome of the major fungal pathogen, Candida albicans, during the heat shock response. The data show that key virulence factors are regulated in response to heat shock, and that Hsp90 exerts major effects on the heat shock transcriptome.a
Project description:To understand how microRNAs are involved in stress response, we examined their expression changes in C. elegans animals that were exposed to stress conditions, including heat shock, oxidation, hypoxia and starvation. Total RNAs were purified from young adult animals that were exposed to each stress, and used for cDNA library preparation for small RNAs. In this experiment, spe-9(hc88), a temperature sensitive sterile mutant, that were cultured at 23dC, was used in order to avoid the effect from developing embryos. Stress conditions we examined include: Heat shock (32°C, 6 hrs), Recovery from heat shock (6 hrs recovery at 23°C after heat shock treatment at 32°C for 6 hrs), Hypoxia (0.01%, 6 hrs), Oxidation (Juglone 750 μM, 6 hrs), Starvation (complete food deprivation, 12 hrs). In addition to these stress conditions, RNAs were prepared from normally cultured, untreated animals at three time points, 0 hr (baseline), 6 hrs (as controls for heat shock, hypoxia and oxidation) and 12 hrs (as controls for heat shock recovery and starvation) after starting stress exposure. These cDNA libraries established were sequenced with Illumina Genome Analyzer II.
Project description:Subspecies of the Atlantic killifish, Fundulus heteroclitus, differ in their maximum thermal tolerance. To determine whether there is a link between the heat shock response (HSR) and maximum thermal tolerance, we exposed 20ºC acclimated killifish from these subspecies to a 2hr heat shock at 34ºC and examined gene expression during heat shock and recovery using real time quantitative PCR and a heterologous cDNA microarray designed for salmonid fishes. Keywords: Expression profiling by array Microarray analyses were performed on four individual fish per subspecies of killifish (northern and southern) prior to heat shock (control) and after 60 minutes of heat shock, hybridized (one slide per individual) against a common reference RNA pool composed of an equal amount of RNA from all samples in the analysis.
Project description:Exiqon miRNA array contains LNA-modified oligos which minimizes the Tm-range within which probe hybridizes with the transcript in query, thus minimizing the mis-match. Taking advantage of this, we used the miRCURY LNA array to investigate genome wide signatures for miRNA expression in response to heat shock stress. We also did mRNA expression profiling using the same RNA to look for possible miRNA-mRNA interaction in response to heat shock stress. For the differentially expressed miRNAs, target predictions were done using both miRanda and TargetScan. The consensus set was used for subsequent experimental validation. In total, 6 comparative hybridizations of heat-shock treated versus control were profiled using the Exiqon arrays. These consisted of 3 biological replicates, each with its dye-swap pair.
Project description:RNA-seq and ribosome footprinting libraries of mouse 3T3 and human 293T cellsrelated to Shalgi et al. 2013 36bases paired-end RNA-seq, and ribosome footprinting libraries for: 3T3 cells - Control, 8 hours of mild heat shock (42) and 2 hours of severe heat shock (44) - in replicates, as well as 3T3 cells treated by mild followed by severe heat shock. In addition, 3T3 cells treated with Hsp70 inhibitor VER-155008 (Massey et al. 2010), and 293T cells transfected with Hspa1a or GFP, before and after 2 hours of severe heat shock.
Project description:Sinorhizobium meliloti can live as a soil saprophyte, and can engage in a nitrogen fixing symbiosis with plant roots. To succeed in such diverse environments, the bacteria must continually adjust gene expression. Transcriptional plasticity in eubacteria is often mediated by alternative sigma factors interacting with core RNA polymerase. The S. meliloti genome encodes 14 of these alternative sigmas, including two putative RpoH (heat shock) sigmas. We used custom Affymetrix Symbiosis Chips to characterize the global transcriptional response of S. meliloti rpoH1, rpoH2 and rpoH1 rpoH2 mutants during heat shock and stationary phase growth. Under these conditions, expression of over 300 genes is dependent on rpoH1 and rpoH2. Gene expression profiling of Sinorhizobium meliloti Rm1021 and its isogenic rpoH1, rpoH2, and rpoH1rpoH2 mutants, subjected to heat shock or stationary phase growth, was performed using custom Affymetrix GeneChips
Project description:To investigate its role during zebrafish development, wnt signalling was either activated by the conditional expression of wnt8b or inhibited by the conditional expression of dickkopf-1b (dkk1b) under the control of the hsp70 heat shock promoter. The transgenic constructs contained a gfp reporter fusion to differentiate between transgenic embryos and their wild type siblings. Transgenic embryos and wild type control embryos at the 48 hours post fertilization stage (hpf) were exposed to heat shock (37 C) for 2 hours and were collected 4 hours after heat shock. The transcriptional response to activated or inhibited wnt signalling was profiled using microarrays and compared to wild type expression.