Studying dynamic transcripts in single cells (SC) requires large numbers of timed samples. We report an easy to use protocol to stabilize RNA in intact SCs without perturbing transcriptional patterns, and demonstrate its applicability for SC transcriptome assays with cells and tissue. We identify a gene-specific hierarchical pattern of all-or-none transcript induction elicited by different concentrations of pulsatile hormone stimuli in pituitary gonadotropes. Overall design: SC Mini Drop-seq experiments were performed on two samples from dissociated human cortical tissue: one is neocortex from a glioblastoma multiforme (GBM, tumor), the other is normal neocortex adjacent to the tumor.
Single-cell stabilization method identifies gonadotrope transcriptional dynamics and pituitary cell type heterogeneity.
Specimen part, Subject
View SamplesWe used microarrays to determine the expression profile of the P. aeruginosa creBC mutant regarding its parental wild type strain PAO1.
The Pseudomonas aeruginosa CreBC two-component system plays a major role in the response to β-lactams, fitness, biofilm growth, and global regulation.
Treatment
View SamplesIn flies, small silencing RNAs are sorted between Argonaute1 (Ago1), the central protein component of the microRNA (miRNA) pathway, and Argonaute2 (Ago2), which mediates RNA interference. Extensive double-stranded character—as is found in small interfering RNAs (siRNAs)—directs duplexes into Ago2, whereas central mismatches, like those found in miRNA/miRNA* duplexes, direct duplexes into Ago1. Central to this sorting decision is the affinity of the small RNA duplex for the Dcr-2/R2D2 heterodimer, which loads small RNAs into Ago2. Here, we show that while most Drosophila miRNAs are bound to Ago1, miRNA* strands accumulate bound to Ago2. Like siRNA loading, efficient loading of miRNA* strands in Ago2 favors duplexes with a paired central region and requires both Dcr-2 and R2D2. Those miRNA and miRNA* sequences bound to Ago2, like siRNAs diced in vivo from long double-stranded RNA, typically begin with cytidine, whereas Ago1-bound miRNA and miRNA* disproportionately begin with uridine. Consequently, some pre-miRNA generate two or more isoforms from the same side of the stem that differentially partition between Ago1 and Ago2. Our findings provide the first genome-wide test for the idea that Drosophila small RNAs are sorted between Ago1 and Ago2 according to their duplex structure and the identity of their first nucleotide. Overall design: Sequencing of small RNAs (either total small RNAs or Ago1-associated small RNAs) in wild-type, dcr-2 and r2d2 mutant flies. Small RNA sequencing, Small RNAs (18-29 nt long), Size selection (18 to 30 nt).
Target RNA-directed trimming and tailing of small silencing RNAs.
Specimen part, Subject
View SamplespiRNA 1U does not cause the secondary piRNA 10A
The initial uridine of primary piRNAs does not create the tenth adenine that Is the hallmark of secondary piRNAs.
Sex, Specimen part, Cell line, Treatment
View SamplesCurrent drugs that directly target pro-angiogenic factors to inhibit or reverse corneal neovascularization, the major sight-threatening pathology caused by angiogenic stimuli, require multiple rounds of administration and have limited efficacies. Here we report the profiling of anti-angiogenic corneal microRNAs (miRNAs), and a framework that employs discovered miRNAs as biotherapies deliverable by recombinant adeno-associated viruses (rAAVs). By querying differentially expressed miRNAs in neovascularized mouse corneas induced by alkali-burn, we have revealed 39 miRNAs that are predicted to target more than 5,500 differentially expressed corneal mRNAs. Among these corneal miRNAs, we selected miR-204 and assessed its efficacy as a therapeutic miRNA in injured corneas. Our results show that delivery of miR-204 by rAAV is efficacious and safe for mitigating corneal NV. Overall, our work demonstrates the discovery of therapeutic miRNAs in corneal disorders and their translation into viable clinical vectors. Overall design: Profiling of mRNAs in normal mouse corneas and corneas injured by alkali burn treatment.
Transcriptome Profiling of Neovascularized Corneas Reveals miR-204 as a Multi-target Biotherapy Deliverable by rAAVs.
Sex, Specimen part, Cell line, Subject
View SamplesGlioblastomas (GBM) are one of the most frequent and aggressive brain tumors. In these malignancies, progesterone (P4) promotes proliferation, migration, and invasion. The P4 metabolite allopregnanolone (3-THP) similarly promotes cell proliferation in the U87 human GBM cell line.
Allopregnanolone Alters the Gene Expression Profile of Human Glioblastoma Cells.
Specimen part, Cell line, Treatment
View SamplesAnimal germ cells produce PIWI-interacting RNAs (piRNAs), small silencing RNAs that suppress transposons and enable gamete maturation. Mammalian transposon-silencing piRNAs accumulate early in spermatogenesis, whereas pachytene piRNAs are produced later during post-natal spermatogenesis and account for >95% of all piRNAs in the adult mouse testis. Mutants defective for pachytene piRNA pathway proteins fail to produce mature sperm, but neither the piRNA precursor transcripts nor the trigger for pachytene piRNA production is known. Here, we show that the transcription factor A-MYB initiates pachytene piRNA production. A-MYB drives transcription of both pachytene piRNA precursor RNAs and the mRNAs for core piRNA biogenesis factors, including MIWI, the protein through which pachytene piRNAs function. A-MYB regulation of piRNA pathway proteins and piRNA genes creates a coherent feed-forward loop that ensures the robust accumulation of pachytene piRNAs. This regulatory circuit, which can be detected in rooster testes, likely predates the divergence of birds and mammals. Overall design: Transcriptome and ChIP sequencing in mouse and rooster testes
An ancient transcription factor initiates the burst of piRNA production during early meiosis in mouse testes.
Specimen part, Cell line, Subject
View SamplesThis SuperSeries is composed of the SubSeries listed below.
A role for microRNAs in the Drosophila circadian clock.
Specimen part, Time
View SamplesWhole-genome expression of peripheral blood leukocytes was measured in 22 patients and 24 controls using the Human Gene 1.0 ST array by Affymetrix
Transcriptomic profile reveals gender-specific molecular mechanisms driving multiple sclerosis progression.
Sex, Age, Specimen part, Disease
View SamplesThis SuperSeries is composed of the SubSeries listed below.
Paternally induced transgenerational environmental reprogramming of metabolic gene expression in mammals.
Sex, Age
View Samples