Project description:We compared the mRNAs expression profile of HeLa cells between two phases of the mitotic cell cycle: S and G2/M phases. Results provide insight into the regulation of transcript levels during mitotic cell cycle progression. HeLa cells were synchronized with double thymidine blockade (12 hours with 2 mM thymidine, 12 hours release, and 12 hours with 2 mM thymidine), and cells were taken after 2 hours release (S phase) and 8 hours release (G2/M phase). Keywords: time course
Project description:We compared the mRNAs expression profile of HeLa cells between two phases of the mitotic cell cycle: S and G2/M phases. Results provide insight into the regulation of transcript levels during mitotic cell cycle progression. HeLa cells were synchronized with double thymidine blockade (12 hours with 2 mM thymidine, 12 hours release, and 12 hours with 2 mM thymidine), and cells were taken after 2 hours release (S phase) and 8 hours release (G2/M phase). Keywords: time course Comparison of mRNAs measured at S phase (2 hours release after double thymidine blockade) and at G2/M phase (8 hours release after double thymidine blockade); 3 biological replicates at each of the two time points; two technical replicates with dye swapping per comparison; additional comparison between G2/M (8h) and S (2h).
Project description:The early transcriptional response of HeLa cervical carcinoma cells to the canonical Wnt signalling pathway was investigated at two different cell cycle phases (G1 and G2/M) via microarray gene expression profiling.
Project description:For the further examination of cell cycle dependent miRNA expression profile, DNA content based fluorescence activated cell sorting (cell cycle sort) was performed on human transformed cancer cell lines. Phase-dependent miRNA expression profiling was performed on G1, S and G2 phases. Results were validated by quantitative real-time PCR. Phase-dependent miRNA expression profiling was performed on sorted human cancer (cervical - HeLa, adrenocortical - NCI-H295R) cells). G1, S and G2 phases were successfully sorted and analyzed.
Project description:For the further examination of cell cycle dependent gene expression profile, DNA content based fluorescence activated cell sorting (cell cycle sort) was performed on human transformed cancer cell lines and primary, untransformed fibroblasts. Phase-dependent gene expression profiling was performed on G1, S and G2 phases. Results were validated by quantitative real-time PCR. Phase-dependent gene expression profiling was performed on sorted human cancer (cervical - HeLa and adrenocortical - NCI-H295R) cells and primary untransformed fibroblasts (HDFa). G1, S and G2 phases were successfully sorted and analyzed. HeLa_mRNA dataset
Project description:The early transcriptional response of HeLa cervical carcinoma cells to the canonical Wnt signalling pathway was investigated at two different cell cycle phases (G1 and G2/M) via microarray gene expression profiling. HeLa cells grown under standard conditions were treated for 3 h with either Control, Wnt3a- or Dkk1-conditioned media and then FACS-sorted into G1 and G2/M populations for microarray expression analysis with 4 independent replicates per group.
Project description:We compared the poly(A) tail length status of mRNAs of HeLa cells between two phases of the mitotic cell cycle: S and G2/M phases. Hundreds of mRNAs were found to be regulated by changes in their poly(A) tail length during mitotic cell cycle in a phase specific manner. Many of these differentially polyadenylated mRNAs encode proteins related to cell death, cell cycle and cellular growth and proliferation. HeLa cells were synchronized with double thymidine blockade (12 hours with 2 mM thymidine, 12 hours release, and 12 hours with 2 mM thymidine), and samples were taken after 2 hours release (S phase) and 8 hours release (G2/M phase). For each condition total RNA was purified by two different procedures: poly(U) chromatography and oligo(dT)-chromatography. Poly(U)-chromatography (Jacobson, 1987): 100 μg of total RNA were bound to poly(U)-sepharose (Sigma) and eluted at 35ºC to isolate mRNAs with short poly(A) tail (<30As, SHORT fraction). Oligo(dT) chromatography: mRNAs were purified independently of their poly(A) tail length with Ambion Poly(A)Purist kit from 20 μg total RNA (ALL fraction). Jacobson, A. Purification and fractionation of poly(A)+ RNA. Methods in Enzymology (1987) 152: 254-261. Keywords: time course
Project description:We compared the poly(A) tail length status of mRNAs of HeLa cells between two phases of the mitotic cell cycle: S and G2/M phases. Hundreds of mRNAs were found to be regulated by changes in their poly(A) tail length during mitotic cell cycle in a phase specific manner. Many of these differentially polyadenylated mRNAs encode proteins related to cell death, cell cycle and cellular growth and proliferation. HeLa cells were synchronized with double thymidine blockade (12 hours with 2 mM thymidine, 12 hours release, and 12 hours with 2 mM thymidine), and samples were taken after 2 hours release (S phase) and 8 hours release (G2/M phase). For each condition total RNA was purified by two different procedures: poly(U) chromatography and oligo(dT)-chromatography. Poly(U)-chromatography (Jacobson, 1987): 100 μg of total RNA were bound to poly(U)-sepharose (Sigma) and eluted at 35ºC to isolate mRNAs with short poly(A) tail (<30As, SHORT fraction). Oligo(dT) chromatography: mRNAs were purified independently of their poly(A) tail length with Ambion Poly(A)Purist kit from 20 μg total RNA (ALL fraction). Jacobson, A. Purification and fractionation of poly(A)+ RNA. Methods in Enzymology (1987) 152: 254-261. Keywords: time course Comparison of ALL fraction mRNAs and SHORT fraction mRNAs measured after 2 hours (S phase) and 8 hours release (G2/M) from double thymidine blockade; 3 biological replicates at each of the two time points; two technical replicates with dye swapping per comparison.
Project description:Gene expression must be reconfigured rapidly during the subsequent phases of the cell cycle to execute the cellular functions specific of each phase. Post-transcriptional regulation has a predominant role in modulating gene expression during the mitotic cell cycle, including among other mechanisms, protein phosphorylation and ubiquitination, differential protein stability and mRNA localization and translatability. Regulation at the transcriptional level is also important, as studies conducted in synchronized plant cell suspension cultures have identified hundreds of genes with periodic patterns of genes expression across the phases of the cell cycle. We describe here an alternative strategy to cell suspension cultures to profile the transcriptome of Arabidopsis root cells in the G2/M phase of the cell cycle. Through fluorescence activated cell sorting we first isolated cells in G2/M using CYCB1;1-GFP, a reporter of a mitotic cyclin. The analysis of the transcriptome of these cells allowed us to identify hundreds of genes whose expression is depleted or enriched in G2/M cells.