RNA-seq transcriptomes of term not in labour and term in labour human myometrial tissue
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ABSTRACT:
ORGANISM(S): Homo sapiens
PROVIDER: GSE50599 | GEO | 2014/02/17
SECONDARY ACCESSION(S): PRJNA218059
REPOSITORIES: GEO
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Project description:Purpose: To chart the human myometrial transcriptomes before and after the onset of labour. Methods: Tophat splice junction mapping of paired-end reads, HTSeq to generate counts, cufflinks to track transcripts, DESeq, edgeR and baySeq to detect differentially expressed genes and principal component analysis for clustering analyses. Results: We mapped on average 14 million paired-end reads per sample (counting each end individually) to the human genome (build hg19) and covered the expressed transcriptome about 13 times with a TopHat-HTSeq workflow. We performed a comparative analysis with an analogous microarray study (Mittal et al., 2010) and found some overlap between the RNA-seq and the microarray data. Conclusions: Our study is the first RNA-seq study of the human myometrium before and after the onset of labour. We show that while microarray and RNA-seq studies may complement each other, RNA-seq has a much greater resolution. At term with and at term without labour human myometrial mRNA profiles were generated by deep sequencing, using Illumina GAIIx (five biological replicates each).
2014-02-17 | E-GEOD-50599 | biostudies-arrayexpress
Project description:Purpose: The cause of labour initiation has yet to be fully elucidated for human pregnancy. This has hindered attempts to find effective therapies for the prevention of preterm labour, which affects up to 10% of pregnancies in the UK and it is the most dominant cause of perinatal death (75% of all cases). The myometrium of the uterus is where contractions that characterise labour take place, and it is here where changes at the molecular level responsible for triggering labour potential originate from. We used RNA-Seq-based mRNA sequencing technology in an attempt to identify mRNA transcripts that are differentially expressed in the myometrium upon the onset of labour, by comparing the expression profiles of tissues samples that represented non-labour (TNL), early labour (TEaL, ≤ 3 cm cervical dilation) and established labour (TEsL, > 3 cm cervical dilation) states at term (> 37 weeks gestation) pregnancy. Methods: Myometrial biopsies from women undergoing Caesarean section were collected in accordance with the Declaration of Helsinki guidelines, and with approval from the local research ethics committee for Chelsea & Westminster Hospital (London, UK; Ethics No. 10/H0801/45). Informed written consent was obtained from all women who participated. Biopsies were excised from the upper margin of the incision made in the lower segment of the uterus, immediately washed with Dulbecco's phosphate-buffered saline (Sigma) and dissected into pieces approximately measuring 2-3 mm3. For RNA study, biopsies were immersed in RNAlater (Sigma) within 6 minutes after biopsy excision from the uterus and stored at 4°c overnight, before being taken out of RNAlater solution to be frozen for long-term storage at -80°c. For CHIP study, biopsies were snap-frozen in liquid nitrogen and stored at -80°c. All specimens were categorised into four groups according to their labour stages: preterm not in labour (PTNL, n=6), term not in labour (TNL, n=8), term in early labour (TEaL, defined as cervical dilatation <3 cm, n=8) and term in established labour (TEsL, defined as cervical dilatation >3 cm, n=6). For each sample, 60-100 mg of myometrium tissue were extracted in TRIzol (Life Technologies) by mechanical homogenisation in a Precellys 24 bead-based homogeniser using 5 cycles of 5000 rpm for 20 seconds, before chloroform treatment and centrifugation at 4°c. RNA was extracted from the aqueous phase of centrifuged homogenates using the TRIzol Plus RNA Purification kit (Life Technologies) with on-column DNase treatment prior to elution, all according to manufacturer's instructions. Final RNA samples were stored at -80°c. The quantity and quality RNA was measured using a Nanodrop ND-1000 spectrophotometer (LabTech), Qubit fluorimeter (Life Technologies) and Bioanalyser 2100 (Agilent Technologies). Preparation of cDNA libraries was carried out using the TruSeq Stranded mRNA Sample Preparation kit (Illumina), following the high-throughput sample (HT) protocol. The quantity and quality of cDNA libraries were also tested by a Qubit fluorimeter and Bioanalyser 2100. TruSeq Stranded libraries were then multiplexed and sequenced with the average of 42 million DNA fragments per sample (100 bp paired-end reads). Quality control was performed using FastQC software (version 0.11.2). RNA-Seq reads was aligned to the GRCh38 Homo sapiens reference genome downloaded from Ensembl (release 81) with HISAT version 2.0.1 using parameters of --known-splicesite-infile --dta-cufflinks --rna-strandness RF --phred33 –p 4 -q. A list of known splice sites generated from the Ensembl GTF file using an accessory python script included in the HISAT2 package was provided to --known-splicesite-infile, of which HISAT2 will make use to assist the alignment of reads spanning two or more exons. Ensembl annotated a total of 65,989 genes, which includes 20,276 protein coding genes. As one human gene usually contains multiple transcript models, we thus conducted a transcript merging procedure to produce gene level models for expression analysis. Specifically, exons labeled as ‘retained_intron’ were first excluded, then overlapping interval exons of each gene were merged and a final gene level model was produced in GFF format. Only uniquely mapped (i.e. reads with the tag of NH:i:1) reads were used to produce gene read counts and calculate gene expression levels. The raw read count matrix was normalised with DESeq2 (version 1.6.3). Expression level of each gene in each sample was represented as RPKM (reads per kilobase per million mapped reads). Differentially expressed genes (DEGs) between two groups of samples were identified with DESeq2 (version 1.6.3), edgeR (version 3.8.6) and Cuffdiff (version 2.2.1). For DESeq2 and edgeR, we used the normalised read count matrix as input, and for Cuffdiff, we used the alignment bam files with uniquely mapped reads as input. Raw p-values were adjusted by FDR to produce q-values, and q-value of 0.05 were chosen as the cut-off for statistical significance in DESeq2, edgeR as well as Cuffdiff. Results: 22 RNA samples from three different labour groups were sequenced and an average of 53 million reads were obtained from each sample. More than 97.34% of reads were successfully aligned to GRCh38 reference human genome and the unique concordant pair ratio was greater than 92.39%. In total, 60,593 genes were mapped with the following criteria: (1) at least one RNA-seq read assigned to a gene; (2) we only assign a read to a gene when > 90% of this read falls into the exon regions of this gene. The principal component analysis (PCA) of these 22 samples showed that TNL and TEsL samples formed two distinct clusters whereas the TEaL group featured relatively great internal differences. Nevertheless, half of the samples from TEaL group was clustered with the TNL group and the other half was more separated yet closer to two samples of TEsL group. To determine the transcripts associated with labour, three software packages (DESeq2, EdgeR and Cuffdiff) were used to perform differential expression The transcript with a q value <0.05 in at least two methods was defined as a shared differentially expressed gene (DEG). As a result, 132 and 399 genes were identified comparing TNL with TEaL and TEsL, respectively, whereas no gene was significantly differentially expressed between TEaL and TEsL groups. Due to big differences among individual samples, in this study, the expression fold change (FC) was calculated as the ratio of median reads per kilobase per million mapped reads (RPKMs) with the bigger median RPKM divided by the smaller median RPKM. In order to minimise the noise derived from genes with low expression but high FC, we further filtered gene lists according to the following rational: if the original value of any median RPKM was <1, we artificially turned it into 1 before calculating the FC. Finally, two robust gene lists with an expression FC >1.5 between two groups (TNL vs. TEaL and TNL vs. TEsL) were generated containing 70 and 232 DEGs, respectively. Conclusions: This study, for the first time, identifies differentially expressed genes (DEGs) and pathways that are involved in the myometrial transition from non-labouring to labouring phenotype by using samples from different stages of pregnancy and labour. The DEG lists are generated by subjecting the raw data to three sofware packages (DESeq2, edgeR and Cuffdiff), which makes the yield DEGs more robust. We conclude that some early responsive genes in circadian clock and inflammation pathways are likely to account for the labour onset and no significant changes are found on the transcription level once the labour starts.
2019-04-23 | GSE80172 | GEO
Project description:Differentially expressed mRNA transcripts in the placenta delivered by term spontaneous labour compared to those delivered by elective term cesarean section. We hypothesized that the labour process involves changes in mRNA expression in the placenta. To test this hypothesis, we interrogated the mRNA levels of >50,000 genes and transcript variants using gene expression microarray (Human Genome U133 Plus 2.0 Array, Affymetrix) on 5 placentas collected from term spontaneous delivery and another 5 placentas collected from elective term cesarean delivery. To minimize the effect of gestational age on gene expression, these two groups of placentas were matched for their gestational ages at delivery. We have identified 134 and 128 genes that were up- or down-regulated, respectively, for more than 3-fold in the term (spontaneous) labour placentas compared to the term (elective) cesarean placentas (Mann-Whitney Rank Sum Test, p-value <= 0.05 after Benjamini and Hochberg adjustment for multiple testing).
2009-11-02 | GSE18850 | GEO
Project description:This study evaluates the gene expression patterns in 36 decidual samples of women having gone through different types of child birth. The samples were profiled on Illumina Human HT-12_v4 BeadArrays. The analysis includes quality control and exploratory analysis of the dataset, followed by the identification of differentially expressed genes (DEGs) and finally, functional analysis for enrichment of KEGG pathways and GO terms amongst the DEGs. The statistical analysis was performed for six comparisons: Term labour (TL) vs Term not labour (TNL), TL vs Preterm labour (PTL), TL vs Preterm non-labour (PTNL), TNL vs PTL, TNL vs PTNL and PTL vs PTNL. This study evaluates the gene expression levels from decidual samples obtained from women giving birth with different types of labour. The dataset was composed of 36 Illumina Human HT-12_v4 expression BeadArrays.
2017-08-23 | E-MTAB-5353 | biostudies-arrayexpress
Project description:Differentially expressed mRNA transcripts in the placenta delivered by term spontaneous labour compared to those delivered by elective term cesarean section. We hypothesized that the labour process involves changes in mRNA expression in the placenta. To test this hypothesis, we interrogated the mRNA levels of >50,000 genes and transcript variants using gene expression microarray (Human Genome U133 Plus 2.0 Array, Affymetrix) on 5 placentas collected from term spontaneous delivery and another 5 placentas collected from elective term cesarean delivery. To minimize the effect of gestational age on gene expression, these two groups of placentas were matched for their gestational ages at delivery. We have identified 134 and 128 genes that were up- or down-regulated, respectively, for more than 3-fold in the term (spontaneous) labour placentas compared to the term (elective) cesarean placentas (Mann-Whitney Rank Sum Test, p-value <= 0.05 after Benjamini and Hochberg adjustment for multiple testing). Experiment Overall Design: Placentas collected from (i) term spontaeous labour and (ii) elective term cesarean section were subjected to RNA extraction and hybridization on Affymetrix microarrays. To identify gene expression patterns that are commonly involved in term spontaneous labour, we analyzed 5 placentas from each of these 2 groups and tested for any differentially expressed genes by non-parametric statistical methods.
2009-11-11 | E-GEOD-18850 | biostudies-arrayexpress
Project description:We have generated genome-wide binding and expression datasets in uterine smooth muscle (myometrium) from murine models of pregnancy and labour. ChIP-seq and total RNA-seq experiments were conducted to evaluate changes in the genomic and transcriptomic landscape of the myometrium prior to, during, and after labour onset.
2020-07-10 | GSE124295 | GEO
Project description:Purpose: The goal of this study is to investigate how Purβ regulates hepatic glucose production in primary hepatocytes. Methods: mRNA profiles of Purβ-overexpressing, and Purβ-knocking down hepatocytes were generated by deep sequencing using an Illumina NovaSeq 6000 platform. Paired-end clean reads were aligned to the mouse reference genome(Ensemble_GRCm38.96) with TopHat (version 2.0.12), and the aligned reads were used to quantify mRNA expression by using HTSeq-count (version 0.6.1). Conclusion: Our study represents the first detailed analysis of mRNA profiles in Purβ-overexpressing, and Purβ-knocking down hepatocytes, generated by RNA-seq technology. Our results identified Adcy6 was a target of Purβ. RNA-seq analysis showed that Purβ overexpression increased Adcy6 expression, whereas Purβ knockdown decreased Adcy6 expression.
2019-09-23 | GSE136728 | GEO
Project description:Biochemistry of spontaneous labour (the second miRNAomic study)
| PRJNA591187 | ENA
Project description:We hypothesized that preterm spontaneous labor involves aberrant changes in mRNA expression in the placenta. To test this hypothesis, we interrogated the mRNA levels of >50,000 genes and transcript variants using gene expression microarray (Human Genome U133 Plus 2.0 Array, Affymetrix) on 5 placentas collected from preterm spontaneous delivery (<34 weeks of gestation) and another 5 placentas collected from term spontaneous delivery (38-39 weeks). We have identified 229 and 162 genes that were up- or down-regulated, respectively, for more than 3-fold in the preterm placentas compared to the term placentas (Mann-Whitney Rank Sum Test, with multiple testing correction by the Benjamini-Hochberg method, adjusted p-value <= 0.05). Placentas collected from (i) preterm spontaneous delivery (<34 weeks of gestation) and (ii) term spontaneous delivery (38-39 weeks of gestation) were subjected to RNA extraction and hybridization on Affymetrix microarrays. To identify gene expression patterns that are commonly involved in preterm spontaneous labour, we analyzed 5 placentas from each of these 2 groups and tested for any differentially expressed genes by Mann-Whitney Rank Sum Test.
2009-10-31 | E-GEOD-18809 | biostudies-arrayexpress
Project description:Sequencing libraries were generated from total RNA samples following the mRNAseq protocol for the generation of single end (16-36 hpf, 5 day larvae, adult head and adult tail) or paired end (24 hpf) libraries (Illumina). Single end reads of 36 nucleotides and paired end reads (2 x 76 nucleotides) were obtained with a GAIIx (Illumina). Gene expression at the different stages/tissu was assessed by cufflinks and HTseq.
2013-07-27 | GSE39703 | GEO