Metabolomics,Unknown,Transcriptomics,Genomics,Proteomics

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MiRNA profiling of H1 ES cells in neural induction timecourse

ABSTRACT: H1 ES cells were differentiated using a neural induction protocol. Biological triplicate samples were collected at five timepoints (d0, d3, d6, d9, d12) and analyzed by miRNA microarrays. The goal of this experiment was to identify potential biases in Drosha processing of miRNAs dependent on biophysical properties and to identify differentially expressed miRNAs over the course of differentiation/neural induction. Using a robust differentiation protocol (described in the associated publication), H1 ES cells underwent differentiation/neural induction for 12 days. Samples were collected in biological triplicate every three days, beginning with day 0 and ending at day 12 for a total of five timpoints and fifteen samples. Total RNA was purified using the Qiagen miRNeasy Mini kit according to the manufacturer and submitted to LC Sciences according to their specifications. Raw data were received and were Lowess-normalized to allow cross-chip comparisons.

ORGANISM(S): Homo sapiens

SUBMITTER: Sherman Ku

PROVIDER: E-GEOD-56107 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:Using criteria based on known examples, we identified approximately 1.7 million and 2.4 million sequences encoding putative miRNA precursor (stem-loop) structures in the mouse and human genomes, respectively, as well as large numbers of such structures in the genomes of fish, fruitfly and nematode worm. These predictions were then tested using high density custom microarrays containing modified oligonucleotides interrogated with purified small RNAs from different tissues. We found that 19% of 4,006 randomly chosen mouse predictions and 40% of 1,859 randomly chosen human predictions gave positive signals with small RNA isolated from whole 10-12 day embryos and a mixture of brain and testis, respectively, 84% of which hybridized to only one strand of the predicted stem-loop structure, whereas only 2% and 12% of equivalent random mouse and human predictions were positive. There was no difference in the validation rates between sequences exhibiting conservation and those that did not. High validation rates were also observed with predictions from fish, fly and worm genomes. Northern blot analysis of the array-positive mouse predictions confirmed that the vast majority of these sequences were detectable as small RNAs whose sizes ranged from 20-110 nucleotides. Taking into account false negative rates of the prediction and detection of known miRNAs, and assuming that this holds for other small RNAs, we estimate that there are 1 million small RNAs expressed in mouse and 3 million small RNAs in human. Keywords: genomic survey of small RNAs Stemloops were identified in the mouse and human genome using RNALfold with a window size of 110nt. Folding energy and other criteria were used to filter the structures into a set of miRNA-like stems. Stems were chosen randomly and probes designed against the stem regions. Stems were called present if one of the stem probes was more than 4 standard deviations above the signal distribution of mRNA degradation contamination probes designed against GAPDH and beta-actin.

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MiRNA profiling of H1 ES cells in neural induction timecourse

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