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Small nuclear RNAs (snRNAs) are

We present primary results from the Sequencing Quality Control (SEQC) project, coordinated by the US Food and Drug Administration. Examining Illumina HiSeq, Life Technologies SOLiD and Roche 454 platforms at multiple laboratory sites using reference RNA samples with built-in controls, we assess RNA sequencing (RNA-seq) performance for junction discovery and differential expression profiling and compare it to microarray and quantitative PCR (qPCR) data using complementary metrics. At all sequencing depths, we discover unannotated exon-exon junctions, with >80% validated by qPCR. We find that measurements of relative expression are accurate and reproducible across sites and platforms if specific-filters are used. In contrast, RNA-seq and microarrays do not provide accurate absolute measurements, and gene-specific biases are observed for all examined platforms, including qPCR. Measurement performance depends on the platform and data analysis pipeline, and variation is large for transcript-level profiling. The complete SEQC data sets, comprising >100 billion reads (10Tb), provide unique resources for evaluating RNA-seq analyses for clinical and regulatory settings.

Gene expression data from microarrays are being applied to predict preclinical and clinical endpoints, but the reliability of these predictions has not been established. In the MAQC-II project, 36 independent teams analyzed six microarray data sets to generate predictive models for classifying a sample with respect to one of 13 endpoints indicative of lung or liver toxicity in rodents, or of breast cancer, multiple myeloma or neuroblastoma in humans. In total, >30,000 models were built using many combinations of analytical methods. The teams generated predictive models without knowing the biological meaning of some of the endpoints and, to mimic clinical reality, tested the models on data that had not been used for training. We found that model performance depended largely on the endpoint and team proficiency and that different approaches generated models of similar performance. The conclusions and recommendations from MAQC-II should be useful for regulatory agencies, study committees and independent investigators that evaluate methods for global gene expression analysis.

RNA polymerase II (Pol II), whose 12 subunits are conserved across eukaryotes, is at the heart of the machinery responsible for transcription of mRNA. Although associated general transcription factors impart promoter specificity, responsiveness to gene- and tissue-selective activators additionally depends on the multiprotein Mediator coactivator complex. We have isolated from tissue extracts a distinct and abundant mammalian Pol II subpopulation that contains an additional tightly associated polypeptide, Gdown1. Our results establish that Gdown1-containing Pol II, designated Pol II(G), is selectively dependent on and responsive to Mediator. Thus, in an in vitro assay with general transcription factors, Pol II lacking Gdown1 displays unfettered levels of activator-dependent transcription in the presence or absence of Mediator. In contrast, Pol II(G) is dramatically less efficient in responding to activators in the absence of Mediator yet is highly and efficiently responsive to activators in the presence of Mediator. Our results reveal a transcriptional control mechanism in which Mediator-dependent regulation is enforced by means of Gdown1, which likely restricts Pol II function only to be reversed by Mediator.

Multipotent stromal cells (MSCs) are being studied in the field of regenerative medicine for their capacity for multi-differentiation. These cells can be isolated from multiple tissue types. The current literature indicates that MSCs have an immunoregulatory capacity, which can suppress the immune system. MicroRNAs (miRNAs) are short non-coding RNAs that are responsible for regulating gene expression. Through targeting binding to gene transcripts, miRNAs have been observed to impact MSC function such as proliferation, differentiation, migration and apoptosis. Studies have shown that various miRNAs are expressed in MSCs; however, the impact of cellular expansion and donor variability on the miRNA expression is not well understood. Six commercially available MSC lines were expanded from passage 3 to 7 and their miRNA expression was evaluated using microarray technology. Statistical analyses of our data revealed that 71 miRNAs out of 939 examined were expressed by this set of MSC lines at all passages and the expression of 13 miRNAs were significantly different between passage 3 and 7. The expression of six miRNAs with the largest fold changes was further evaluated using RT-qPCR for both the original MSC lines and a second set of seven MSC lines expanded from passage 4 to 8. By RT-qPCR only 2 miRNAs, miR-638 and miR-572 were upregulated at passage 7 compared to passage 3 for the original MSC lines by 1.71 and 1.54 fold, respectively; and upregulated at passage 8 compared to passage 4 for the second set of MSC lines, 1.34 and 1.59 fold, respectively. These 2 miRNAs distinguish aging MSCs expanded in culture. These novel results may be useful in establishing critical quality attributes for limiting clinical applications of MSCs beyond specific cellular expansion protocols.

BackgroundA common element among cancer cells is the presence of improperly controlled transcription. In these cells, the degree of specific activation of some genes is abnormal, and altering the aberrant transcription may therefore directly target cancer. TFIIS is a transcription elongation factor, which directly binds the transcription motor, RNA Polymerase II and allows it to read through various transcription arrest sites. We report on RNA interference of TFIIS, a transcription elongation factor, and its affect on proliferation of cancer cells in culture.MethodsRNA interference was performed by transfecting siRNA to specifically knock down TFIIS expression in MCF7, MCF10A, PL45 and A549 cells. Levels of TFIIS expression were determined by the Quantigene method, and relative protein levels of TFIIS, c-myc and p53 were determined by C-ELISA. Induction of apoptosis was determined by an enzymatic Caspase 3/7 assay, as well as a non-enzymatic assay detecting cytoplasmic mono- and oligonucleosomes. A gene array analysis was conducted for effects of TFIIS siRNA on MCF7 and MCF10A cell lines.ResultsKnockdown of TFIIS reduced cancer cell proliferation in breast, lung and pancreatic cancer cell lines. More specifically, TFIIS knockdown in the MCF7 breast cancer cell line induced cancer cell death and increased c-myc and p53 expression whereas TFIIS knockdown in the non-cancerous breast cell line MCF10A was less affected. Differential effects of TFIIS knockdown in MCF7 and MCF10A cells included the estrogenic, c-myc and p53 pathways, as observed by C-ELISA and gene array, and were likely involved in MCF7 cell-death.ConclusionAlthough transcription is a fundamental process, targeting select core transcription factors may provide for a new and potent avenue for cancer therapeutics. In the present study, knockdown of TFIIS inhibited cancer cell proliferation, suggesting that TFIIS could be studied as a potential cancer target within the transcription machinery.

One common way to examine toxicity in vitro is to measure the effect on cell viability. In one such assay, the 3-(4,5- dimethylthiazol-2-yl)-5-(3 -carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt MTS reagent is bioreduced to a formazan product in living cells. Various cell lines may have differing abilities to reduce MTS; therefore, standardization should be carried out for each one. To optimize the assay, the toxicity of MTS, the linear range for signal versus cell number, and a method of background noise reduction were determined. The MTS reagent decreased cell number after 25 hr. The linear range for the neuronal SK-N-Sll, esophageal KYSE-30, breast MCF-7, and cervical HeLa cell lines were established. Finally, media containing no phenol red significantly reduced background noise compared to media with phenol red.

Introduction Human multipotent stromal cells (MSCs) isolated from bone marrow or other tissue sources have great potential to treat a wide range of injuries and disorders in the field of regenerative medicine and tissue engineering. In particular, MSCs have inherent characteristics to suppress the immune system and are being studied in clinical studies to prevent graft-versus-host disease. MSCs can be expanded in vitro and have potential for differentiation into multiple cell lineages. However, the impact of cell passaging on gene expression and function of the cells has not been determined. Methods Commercially available human MSCs derived from bone marrow from six different donors, grown under identical culture conditions and harvested at cell passages 3, 5, and 7, were analyzed with gene-expression profiling by using microarray technology. Results The phenotype of these cells did not change as reported previously; however, a statistical analysis revealed a set of 78 significant genes that were distinguishable in expression between passages 3 and 7. None of these significant genes corresponded to the markers established by the International Society for Cellular Therapy (ISCT) for MSC identification. When the significant gene lists were analyzed through pathway analysis, these genes were involved in the top-scoring networks of cellular growth and proliferation and cellular development. A meta-analysis of the literature for significant genes revealed that the MSCs seem to be undergoing differentiation into a senescent cell type when cultured extensively. Consistent with the differences in gene expression at passage 3 and 7, MSCs exhibited a significantly greater potential for cell division at passage 3 in comparison to passage 7. Conclusions Our results identified specific gene markers that distinguish aging MSCs grown in cell culture. Confirmatory studies are needed to correlate these molecular markers with biologic attributes that may facilitate the development of assays to test the quality of MSCs before clinical use.