cDNA Labeling Research Articles

Gene Expression Profiling during Life Cycle of Potato (Solanum tuberosum) Tubers by Microarray

Microarray was used to investigate the gene expression profile of potato tubers during its complete life cycle. The potato life cycle was divided into 8 different stages and total RNA was isolated and used as probe after labeling of cDNA generated from RNA samples. cDNAs were used to hybridize with potato microarray. Results of this study revealed that there is upregulation of many genes during developing stages (3b, 3c, 3d, 4a, 4b) compared to mature stages (6, 7). Many of those genes regulate growth, storage of protein and sugar, defense, inhibition of various proteinases, and oxidation. Some genes showed upregulation during certain growth stage (4a) compared to other growth stages, whereas, other genes showed upregulation in mature stages (stage 8). Profiling of gene expression during potato life cycle is essential step for enhancing potato for some highly economic traits like tuber nutrition quality, resistance to diseases, or using tubers as source for proteinase inhibitors. Also, this allows for isolation of some genes for biotechnology applications like proteinase inhibitors and improving nutrition and industrial value of tubers.

Pathway analysis of differentially expressed genes in Mycobacterium bovis challenged bovine macrophages

The immune signalling genes during the challenge of bovine macrophages with bacterial products derived from tuberculosis causing bacteria in cattle were investigated in the present study. An in-vitro cell culture model of bovine monocyte-derived macrophages were challenged to Mycobacterium bovis. Macrophages from healthy and already infected animals can both be fully activated during M. bovis infection. Analysis of mRNA abundance in peripheral blood mononuclear cells from M. bovis infected and non-infected cattle were performed as a controls. Cells of treatment were challenged after six days for six hours incubation at 37 °C, with 5% CO2, to total RNA was extracted then cDNA labelling, hybridization and scanning for microarray methods have been developed for microarray based immune related gene expression analysis. The differential expressions twenty genes (IL1, CCL3, CXCR4, TNF, TLR2, IL12, CSF3, CCR5, CCR3, MAPT, NFKB1, CCL4, IL6, IL2, IL23A, CCL20, IL8, CXCL8, TRIP10, CXCL2 and IL1B) implicated in M. bovis response were examined Agilent Bovine_GXP_8 × 60 K microarray platform. Cells of treatment were challenged after six days for six hours incubation then pathways analysis of Toll like receptor and Chemokine signalling pathway study of responsible genes in bovine tuberculosis. The PBMC from M. bovis infected cattle exhibit different transcriptional profiles compared with PBMC from healthy control animals in response to M. bovis antigen stimulation, providing evidence of a novel genes expression program due to M. bovis exposure. It will guide future studies, regarding the complex macrophage specific signalling pathways stimulated upon phagocytosis of M. bovis and role of signalling pathways in creating the host immune response to cattle tuberculosis.

Hindlimb Muscles of 17.5–18.5 dpc Mice Double Null for MyoD and Trp53 Appear Indistinguishable from Muscles of Mice Null for Either Gene

It has been shown by others that mice with either a MyoD null (MN) or Trp53 null (PN) genotype undergo myogenesis resulting in perinatal hindlimb muscle essentially indistinguishable from that of wild type (WT), while myogenic cells of the same null genotypes undergo impaired differentiation in culture. Both MyoD and Trp53 act through p21 and pRb to influence cell cycle withdrawal and differentiation. We hypothesized that redundancies in the activities of Trp53 and MyoD might rescue in vivo myogenesis in animals null for either protein, in which case animals double null (DN) for MyoD and Trp53 should demonstrate impaired hindlimb myogenesis. We bred PN and MN animals and their progeny to obtain WT, MN, PN, and DN pups. At 17.5–18.5 dpc, all possible genotypes, including DN, and both genders were present with no significant difference from the expected Mendelian distributions. Survival to adulthood was significantly reduced among MN and DN animals (P<0.001), with reduction in DN more severe. The effect of PN on survival was unclear. Location, shapes, and domains of myonuclei and alignment, continuity, branching pattern, and number and diameter of the muscle fibers were compared between MN and DN siblings, using paraffin‐embedded, hematoxylin and eosin‐stained sections of extensor digitorum longus (EDL) fast‐twitch and soleus (SOL) slow‐twitch muscles. DN muscles had no detectable deficiencies compared to the muscles of MN (N = 7 sibling sets of each muscle type). mRNA levels were compared across all 4 genotypes using NimbleGen Systems 60‐mer expression arrays, 2 arrays per genotype. Two biologically independent cDNA samples were prepared per genotype, each from pools of RNA isolated from hindlimb muscle of 5–6 mice. cDNA labeling, array hybridization, and initial normalization of data were carried out by Nimblegen. Subsequent analysis using ArrayStar software required substantial accommodation for scatter. 4417 calls (~2873 genes) with >2‐fold difference in expression level between the two arrays of any one genotype were removed from analysis and the remaining 38,169 calls (~24,831 genes) were rescaled by quantile ranking. Of genes eliminated, ~135 were involved in muscle function (Panther Database, PD), but only 2 of the 135 were eliminated because of scatter unique to the DN arrays. Thus increased scatter of muscle gene expression cannot be attributed specifically to the DN genotype. Four genes used for normalization of mRNA studies in muscle, Hprt1, Gapdh, Sf3a1, and Hadha, showed consistent levels of expression across all 4 genotypes. mRNAs from the different members of the myosin heavy chain gene family showed the expected diversity in levels of expression. Only 7 genes passed our multi‐step screening for differential expression with a >2.2 fold difference in expression between any 2 genotypes, including VIP neuropeptide hormone, and Myosin heavy chain 4 (Myh4), both involved in muscle contraction. Myh4 mRNA was elevated in MN and DN mice. Vip mRNA was elevated in the DN relative to PN but less relative to MN and WT. Thus, we found no convincing support for a model in which p53 and MyoD act as back‐ups for each other during prenatal myogenesis. It would be of interest to determine the effect of the DN on muscle regeneration and neuromuscular development, which have been shown to be abnormal in the MN mouse. Animal studies were performed in accordance with Cal State LA IACUC 06‐5.Support or Funding InformationCal State LA MBRS‐RISE 1 R25 GM61331 to C. Gutierrez; NIH‐AREA Grant #R15 AR51322‐01 to Sandra B. Sharp

PD33-11 NEOADJUVANT CHEMOTHERAPY IN BLADDER CANCER: P53-NESS IS ASSOCIATED WITH CHEMO-RESISTANCE AND UNFAVORABLE OUTCOME

You have accessJournal of UrologyBladder Cancer: Invasive IV1 Apr 2016PD33-11 NEOADJUVANT CHEMOTHERAPY IN BLADDER CANCER: P53-NESS IS ASSOCIATED WITH CHEMO-RESISTANCE AND UNFAVORABLE OUTCOME Roland Seiler, Woonyoung Choi, Nicholas Erho, Bernhard Kiss, Lucia L Lam, Qici Wang, Christine Bürki, Elai Davicioni, George N Thalmann, David J McConkey, and Peter C Black Roland SeilerRoland Seiler More articles by this author , Woonyoung ChoiWoonyoung Choi More articles by this author , Nicholas ErhoNicholas Erho More articles by this author , Bernhard KissBernhard Kiss More articles by this author , Lucia L LamLucia L Lam More articles by this author , Qici WangQici Wang More articles by this author , Christine BürkiChristine Bürki More articles by this author , Elai DavicioniElai Davicioni More articles by this author , George N ThalmannGeorge N Thalmann More articles by this author , David J McConkeyDavid J McConkey More articles by this author , and Peter C BlackPeter C Black More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2016.02.720AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Muscle-invasive bladder cancer (MIBC) can molecularly be grouped into intrinsic basal and luminal subtypes. Tumors within the luminal subtype and a ′p53-like′ gene expression have been found to be resistant to neoadjuvant chemotherapy (NAC) with methotrexate, vinblastine, doxorubicin, and cisplatin (MVAC). We investigated the response of these subtypes to gemcitabine plus cisplatin (GC), the other major frontline NAC regimen in this disease setting. METHODS Fifty-two MIBC patients received neoadjuvant GC followed by cystectomy. At cystectomy, 37 (71%) patients did not respond to NAC (ypT≥2 or any ypN1-3). With the RNeasy FFPE kit (Qiagen) RNA was isolated from pre-NAC transurethral resection (TUR) specimens and post-NAC cystectomy specimens. After cDNA ampliciation and labelling with the Ovation WTA FFPE system and Encore Biotin Module (NuGen) samples were hybridized to GeneChip Human Exon 1.0 ST oligonucleotide microarrays (Affymetrix). The tumors were assigned to the intrinsic subtypes using a one nearest neighbor prediction model. RESULTS Unsupervised hierarchical clustering separated the tumors into clusters characterized by non-overlapping expression of basal or luminal biomarkers. Assignment of pretreatment TUR tumors to subtypes yielded the expected ratios of basal, p53-like, and luminal tumors of which 5/14 (36%), 2/15 (17%) and 8/23 (35%) showed pathological down staging (<ypT2 any ypN neg), respectively. p53-like tumors had significantly the shortest recurrence free (p=0.006) and overall survival (p=0.003) compared to the other subtypes. Basal tumors with an enrichment of an immune signature responded to NAC, although this did not reach statistical significance. Matched analysis of tumors before and after NAC revealed an enrichment for p53-like tumors at cystectomy. CONCLUSIONS In line with the MVAC data, p53-like tumors displayed resistance to neoadjuvant GC. Moreover, p53-ness is associated with unfavorable outcome in MIBC treated with NAC. If these results are confirmed prospectively, patients with p53-like tumors should not be treated with cisplatin-based chemotherapy and should instead be steered to immediate cystectomy or clinical trials of novel therapies. © 2016FiguresReferencesRelatedDetails Volume 195Issue 4SApril 2016Page: e770 Advertisement Copyright & Permissions© 2016MetricsAuthor Information Roland Seiler More articles by this author Woonyoung Choi More articles by this author Nicholas Erho More articles by this author Bernhard Kiss More articles by this author Lucia L Lam More articles by this author Qici Wang More articles by this author Christine Bürki More articles by this author Elai Davicioni More articles by this author George N Thalmann More articles by this author David J McConkey More articles by this author Peter C Black More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

From RNA isolation to microarray analysis: Comparison of methods in FFPE tissues

BackgroundGenome-wide gene expression profiling analysis of FFPE tissue samples is indispensable for cancer research and provides the opportunity to evaluate links between molecular and clinical information, however, working with FFPE samples is challenging due to extensive cross-linking, fragmentation and limited quantities of nucleic acid. Thus, processing of FFPE tissue samples from RNA extraction to microarray analysis still needs optimization. Materials and methodsIn this study, a modified deparaffinization protocol was conducted prior to RNA isolation. Trizol, Qiagen RNeasy FFPE and Arcturus PicoPure RNA Isolation kits were used in parallel to compare their impact on RNA isolation. We also evaluated the effect of two different cRNA/cDNA preparation and labeling protocols with two different array platforms (Affymetrix Human Genome U133 Plus 2.0 and U133_X3P) on the percentage of present calls. ResultsOur optimization study shows that the Qiagen RNeasy FFPE kit with modified deparaffinization step gives better results (RNA quantity and quality) than the other two isolation kits. The Ribo-SPIA protocol gave a significantly higher percentage of present calls than the 3′ IVT cDNA amplification and labeling system. However, no significant differences were found between the two array platforms. ConclusionOur study paves the way for future high-throughput transcriptional analysis by optimizing FFPE tissue sample processing from RNA isolation to microarray analysis.

Uncultured Desulfobacteraceae and Crenarchaeotal group C3 incorporate 13C-acetate in coastal marine sediment.

Stable isotope probing (SIP) of deoxyribonucleic acid (DNA) was used to identify microbes incorporating (13) C-labeled acetate in sulfate-reducing sediment from Aarhus Bay, Denmark. Sediment was incubated in medium containing 10 mM sulfate and different (13) C-acetate (10, 1, 0.1 mM) concentrations. The resultant changes in microbial community composition were monitored in total and SIP-fractionated DNA during long-term incubations. Chemical analyses demonstrated metabolic activity in all sediment slurries, with sulfate-reducing activity largely determined by initial acetate concentrations. Sequencing of 16S rRNA gene PCR amplicons showed that the incubations shifted the bacterial but not the archaeal community composition. After 3 months of incubation, only sediment slurries incubated with 10 mM (13) C-acetate showed detectable (13) C-DNA labeling. Based on 16S rRNA and dsrB gene PCR amplicon sequencing, the (13) C-labeled DNA pool was dominated by a single type of sulfate reducer representing a novel genus in the family Desulfobacteraceae. In addition, members of the uncultivated Crenarchaeotal group C3 were enriched in the (13) C-labeled DNA. Our results were reproducible across biological replicate experiments and provide new information about the identities of uncultured acetate-consuming bacteria and archaea in marine sediments.

Bufalin alters gene expressions associated DNA damage, cell cycle, and apoptosis in human lung cancer NCI-H460 cells in vitro.

Lung cancer is the leading cause of cancer related death and there is no effective treatment to date. Bufalin has been shown effective in inducing apoptosis and DNA damage in lung cancer cells. However, the genetic mechanisms underlying these actions have not been elucidated yet. Cultured NCI-H460 cells were treated with or without 2 μM of bufalin for 24 h. The total RNA was extracted from each treatment for cDNA synthesis and labeling, microarray hybridization, and then followed by flour-labeled cDNA hybridized on chip. The localized concentrations of fluorescent molecules were detected and quantitated and analyzed by Expression Console software (Affymetrix) with default RMA parameters. The key genes involved and their possible interaction pathways were mapped by GeneGo software. About 165 apoptosis-related genes were affected. CASP9 was up-regulated by 5.51 fold and THAP1 by 2.75-fold while CCAR1 was down-regulated by 2.24 fold. 107 genes related to DNA damage/repair were affected. MDC1 was down-regulated by 2.22-fold, DDIT4 by 2.52 fold while GADD45B up-regulated by 3.72 fold. 201 genes related to cell cycles were affected. CCPG1 was down-regulated by 2.11 fold and CDCA7L by 2.71 fold. Many genes about apoptosis, cell cycle regulation and DNA repair are changed significantly following bufalin treatment in NCI-H460 cells. These changes provide an in depth understanding of cytotoxic mechanism of bufalin in genetic level and also offer many potentially useful biomarkers for diagnosis and treatment of lung cancer in future.

AA-Dutp-Cy3 a Novel Fluorescent Labeling Agent for Nucleotides

A bioassay was developed to provide a process for the synthesis of cyanine dye labeled base modified nucleoside triphosphates analogues as a novel labeling agent for biomolecules. It is also to provide a low cost process and more specifically relates to a process for the synthesis of cyanine fluorophore - base modified nucleoside triphosphates analogues as well as for efficient and robust labeling of RNA and cDNA as hybridization probe in microarray detection and analysis.

The ATP-dependent Chromatin Remodeling Enzyme Fun30 Represses Transcription by Sliding Promoter-proximal Nucleosomes

The evolutionarily conserved ATP-dependent chromatin remodeling enzyme Fun30 has recently been shown to play important roles in heterochromatin silencing and DNA repair. However, how Fun30 remodels nucleosomes is not clear. Here we report a nucleosome sliding activity of Fun30 and its role in transcriptional repression. We observed that Fun30 repressed the expression of genes involved in amino acid and carbohydrate metabolism, the stress response, and meiosis. In addition, Fun30 was localized at the 5' and 3' ends of genes and within the open reading frames of its targets. Consistent with its role in gene repression, we observed that Fun30 target genes lacked histone modifications often associated with gene activation and showed an increased level of ubiquitinated histone H2B. Furthermore, a genome-wide nucleosome mapping analysis revealed that the length of the nucleosome-free region at the 5' end of a subset of genes was changed in Fun30-depleted cells. In addition, the positions of the -1, +2, and +3 nucleosomes at the 5' end of target genes were shifted significantly, whereas the position of the +1 nucleosome remained largely unchanged in the fun30Δ mutant. Finally, we demonstrated that affinity-purified, single-component Fun30 exhibited a nucleosome sliding activity in an ATP-dependent manner. These results define a role for Fun30 in the regulation of transcription and indicate that Fun30 remodels chromatin at the 5' end of genes by sliding promoter-proximal nucleosomes.

Uncovering Molecular Events Associated with Chemosuppressant Effects of Flaxseed: A Microarray Analysis in Laying Hen Model of Ovarian Cancer.

Despite considerable efforts to improve early detection and advances in therapy, ovarian cancer remains a major challenge. Significant lack of early-stage malignancy symptoms and evidence of disease after metastasis; contribute to very low survival rate in patients. Resistance to available chemotherapeutic drug regimes and tumor relapse added much to the lethality of the disease. These limitations encouraged us to search for other strategies for management of the disease. The activity of Omega-3 fatty acid reported against cancer, therefore it may be a safe agent to reduce the progression of ovarian cancer. A study was conducted to evaluate the efficacy of flaxseed (richest plant source of Omega-3 fatty acid) on laying hen an excellent animal model for ovarian cancer. Three hundred eighty-seven single-comb 2.5-year-old hens were randomly divided into Control and Flax groups. Control groups hens were subdivided into Control Normal and Control Cancer, whereas Flax group in Flax Normal and Flax Cancer. Control hens were fed with standard diet, while Flax was with diet supplemented with 10% flaxseed. Results show a significant reduction in the severity of the disease and increased survival of the laying hens fed with flaxseed. Moving further, we tried to elucidate the molecular mechanism underlying the tumor suppressive property of flaxseed. We used Agilent custom 4x44K chicken long oligo microarray, designed by Dr. Zhou of Texas A&M University. Routine RNA extraction and precipitation were performed from ovary tissue of 4 groups of hens. Samples were sent to the WM Keck center for Comparative & Functional Genomics, University of Illinois Urbana-Champaign for cDNA synthesis, labeling, hybridization, scanning and preliminary analysis. Microarray profiling demonstrated around 43,803 genes which have higher intensity of fluorescence after normalization against background. We applied 1.5 fold change cutoffs i.e. all genes expressing intensity of high or lower than 1.5 fold changes were only selected, this effort generated a reasonable numbers of genes for further analysis. We performed 6 way pair analysis of the gene list between the groups i.e. 1.Control Cancer to Control Norm; 2.Control Cancer to Flax Normal; 3. Flax Normal to Flax Cancer;4. Flax Cancer to Control Normal;5. Control Normal to Flax Normal and 6.Flax Cancer to Control Cancer which suggested significant alteration in expression pattern of genes between the sample groups. We applied various bioinformatics tools such as Multiple Experimental Viewer, Ingenuity Pathway Analysis and Gene Ontology Enrichment Analysis Software Tools for analysis. Microarray analysis identified genes were associated with key molecular and cellular functions such as- cell survival, cell Cycle, molecular mechanisms of cancer, cell-to-cell signaling and cellular growth & proliferation. The top signaling pathways appeared on analyses were p53 signaling, FAK signaling, Fructose metabolism and EGF signaling. Representative important molecules were evaluated by RT-PCR and their expression profile matched with the microarray result. Altogether these data suggested that activated cell survival, cell cycle and p53 signaling may contribute to the reduction in the severity of disease and increased cell survival in hens. This study is an increment towards understanding the molecular mechanism underlying the effect of flaxseed rich diet in laying hen model of ovarian cancer. Accordingly, natural products such as flaxseed may be promising agent for dietary intervention in ovarian cancer-therapy. [Supported by NIH/NCCAM AT004085 and NCI CA133915] (poster)

Abstract 732: Characterization of gene expression changes associated with Erlotinib and Temsirolimus treatment in glioma cell lines

Abstract Background: The use of tyrosine kinase inhibitors as erlotinib (ERL) has been widely studied for the treatment of human tumors including malignant glioma. To enhance the effect of ERL, coadyuvant treatment with an inhibitor downstream the EGFR pathway, such as temsirolimus (TEMS), has been proposed (Wang et al, 2006). Here, we analyze the gene expression profiles of a large series of human glioma cell lines with different sensitivity to ERL and TEMS, to identify the molecular effects of both inhibitors. Materials and methods: Eleven glioma cell lines (U373, U87MG, U118, SW1088, A172, SW1783, H4, GOS3, LN18, SF767, T98G) where treated for 24 hours in a serum-starved medium with 10µM ERL alone or in combination with 0,01µM TEMS. Both treatments and basal cultures were stimulated with 50ng/ml EGF during treatment. Total RNAs were trizol extracted. After cDNA synthesis and labeling, hybridations were made on Agilent's Whole Human Genome expression arrays that consist of about 40,000 genes. Statistical analyses were done with GEPAS and Babelomics. Results: The samples, treated with ERL, ELR+TEMS or untreated, significantly grouped by cell line independently of treatment. There are 52 differentially expressed genes (FDR&amp;lt;0,15) between treated and untreated cell lines. Among them, Gas5, which promotes apoptosis, raised its expression in treated cell lines, while PLK1 and BIRC5, that activates cell cycle and inhibits apoptosis, respectively, diminished its expression with treatment (FDR&amp;lt;0.05). Analyses of gene ontologies (GO) showed 3 BIOCARTA pathways overexpressed before treatment in these cell lines (p&amp;lt;0,05). Two of them related to cell cycle control (Ran and Akap95 pathways) and one to invasiveness and metastatis (Mta3 pathway). Besides, we compared cell lines with different sensitivity to erlotinib (8 resistant and 3 sensitive, as characterized by viability assays) in order to identify genes responsible of the different response to ERL. Among the 1,191 significant deregulated genes (FDR&amp;lt;0.15), we observed HRAS overexpression in resistant cell lines. In addition, 29 transcription factors were diferentially expressed (p&amp;lt;0,05). Remarkably, Hes-1 also overexpressed in resistant cell lines, is involved in neurogenesis and keeps cells indiferenciated and in proliferation. Conclusion: Although further confirmation of these results is needed, we have found that treatment of cell lines with ERL and ERL+TEMS affect cell cycle and cell growth. In addition, we have identify genes that could be related to the diferential response to ERL treatment in glioma cell lines, such as Hras and Hes-1. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 732. doi:10.1158/1538-7445.AM2011-732

Growth and Metastases of Human Lung Cancer Are Inhibited in Mouse Xenografts by a Transition State Analogue of 5′-Methylthioadenosine Phosphorylase

The S-adenosylmethionine (AdoMet) salvage enzyme 5'-methylthioadenosine phosphorylase (MTAP) has been implicated as both a cancer target and a tumor suppressor. We tested these hypotheses in mouse xenografts of human lung cancers. AdoMet recycling from 5'-methylthioadenosine (MTA) was blocked by inhibition of MTAP with methylthio-DADMe-Immucillin-A (MTDIA), an orally available, nontoxic, picomolar transition state analogue. Blood, urine, and tumor levels of MTA increased in response to MTDIA treatment. MTDIA treatment inhibited A549 (human non-small cell lung carcinoma) and H358 (human bronchioloalveolar non-small cell lung carcinoma cells) xenograft tumor growth in immunodeficient Rag2(-/-)γC(-/-) and NCr-nu mice. Systemic MTA accumulation is implicated as the tumor-suppressive metabolite because MTDIA is effective for in vivo treatment of A549 MTAP(-/-) and H358 MTAP(+/+) tumors. Tumors from treated mice showed increased MTA and decreased polyamines but little alteration in AdoMet, methionine, or adenine levels. Gene expression profiles of A549 tumors from treated and untreated mice revealed only modest alterations with 62 up-regulated and 63 down-regulated mRNAs (≥ 3-fold). MTDIA antitumor activity in xenografts supports MTAP as a target for lung cancer therapy.

Comparison of transcriptional and translational changes caused by long-term menadione exposure in Aspergillus nidulans

Under long-term oxidative stress caused by menadione sodium bisulfite, genome-wide transcriptional and proteome-wide translational changes were compared in Aspergillus nidulans vegetative cells. The comparison of proteomic and DNA microarray expression data demonstrated that global gene expression changes recorded with either flip-flop or dendrimer cDNA labeling techniques supported proteome changes moderately with 40% and 34% coincidence coefficients, respectively. Enzyme levels in the glycolytic pathway were alternating, which was a direct consequence of fluctuating gene expression patterns. Surprisingly, enzymes in the vitamin B2 and B6 biosynthetic pathways were repressed concomitantly with the repression of some protein folding chaperones and nuclear transport elements. Under long-term oxidative stress, the peroxide-detoxifying peroxiredoxins and cytochrome c peroxidase were replaced by thioredoxin reductase, a nitroreductase and a flavohemoprotein, and protein degradation became predominant to eliminate damaged proteins.

395 Valid PCR quantification of mRNA from 16 year old formalin-fixed, paraffin-embedded breast cancer tissue: a methodological study comparing manually trimmed sections and whole tissue sections

Genome-wide gene expression profiling analysis of FFPE tissue samples is indispensable for cancer research and provides the opportunity to evaluate links between molecular and clinical information, however, working with FFPE samples is challenging due to extensive cross-linking, fragmentation and limited quantities of nucleic acid. Thus, processing of FFPE tissue samples from RNA extraction to microarray analysis still needs optimization.In this study, a modified deparaffinization protocol was conducted prior to RNA isolation. Trizol, Qiagen RNeasy FFPE and Arcturus PicoPure RNA Isolation kits were used in parallel to compare their impact on RNA isolation. We also evaluated the effect of two different cRNA/cDNA preparation and labeling protocols with two different array platforms (Affymetrix Human Genome U133 Plus 2.0 and U133_X3P) on the percentage of present calls.Our optimization study shows that the Qiagen RNeasy FFPE kit with modified deparaffinization step gives better results (RNA quantity and quality) than the other two isolation kits. The Ribo-SPIA protocol gave a significantly higher percentage of present calls than the 3′ IVT cDNA amplification and labeling system. However, no significant differences were found between the two array platforms.Our study paves the way for future high-throughput transcriptional analysis by optimizing FFPE tissue sample processing from RNA isolation to microarray analysis.

Brain-Specific Deletion of Extracellular Signal-Regulated Kinase 2 Mitogen-Activated Protein Kinase Leads to Aberrant Cortical Collagen Deposition

The mitogen-activated protein kinases extracellular signal-regulated kinase (ERK)1 and 2 are essential intracellular mediators of numerous transmembrane signals. To investigate neural-specific functions of ERK2 in the brain, we used a Cre/lox strategy using Nestin:Cre to drive recombination in neural precursor cells. Nestin:Cre;ERK2(fl/fl) conditional knockout (cKO) mice have architecturally normal brains and no gross behavioral deficits. However, all cKO mice developed early-onset (postnatal day 35 to 40) frontal cortical astrogliosis, without evidence of neuronal degeneration. Frontoparietal cortical gray matter, but not underlying white matter, was found to contain abundant pericapillary and parenchymal reticulin fibrils, which were shown by immunohistochemistry to contain fibrillar collagens, including type I collagen. ERK1 general KO mice showed neither fibrils nor astrogliosis, indicating a specific role for ERK2 in the regulation of brain collagen. Collagen fibrils were also observed to a lesser extent in GFAP:Cre;ERK2(fl/fl) mice but not in CamKII-Cre;ERK2(fl/fl) mice (pyramidal neuron specific), consistent with a possible astroglial origin. Primary astroglial cultures from cKO mice expressed elevated fibrillar collagen levels, providing further evidence that the phenotype may be cell autonomous for astroglia. Unlike most other tissues, brain and spinal cord parenchyma do not normally contain fibrillar collagens, except in disease states. Determining mechanisms of ERK2-mediated collagen regulation may enable targeted suppression of glial scar formation in diverse neurological disorders.

A rapid and inexpensive labeling method for microarray gene expression analysis

BackgroundGlobal gene expression profiling by DNA microarrays is an invaluable tool in biological research. However, existing labeling methods are time consuming and costly and therefore often limit the scale of microarray experiments and sample throughput. Here we introduce a new, fast, inexpensive method for direct random-primed fluorescent labeling of eukaryotic cDNA for gene expression analysis and compare the results obtained on the NimbleGen microarray platform with two other widely-used labeling methods, namely the NimbleGen-recommended double-stranded cDNA protocol and the indirect (aminoallyl) method.ResultsTwo total RNA samples were labeled with each method and hybridized to NimbleGen expression arrays. Although all methods tested here provided similar global results and biological conclusions, the new direct random-primed cDNA labeling method provided slightly better correlation between replicates compared to the other methods and thus increased ability to find statistically significant differentially expressed genes.ConclusionThe new direct random-primed cDNA labeling method introduced here is suitable for gene expression microarrays and provides a rapid, inexpensive alternative to existing methods. Using NimbleGen microarrays, the method produced excellent results comparable to those obtained with other methods. However, the simplicity and cost-effectiveness of the new method allows for increased sample throughput in microarray experiments and makes the process amenable to automation with a relatively simple liquid handling system.

Reduced Expression of an RNA-binding Protein by Prolactin Leads to Translational Silencing of Programmed Cell Death Protein 4 and Apoptosis in Newt Spermatogonia

Recent studies indicate that the balance between cell survival and proapoptotic signals determines which cells commit to life or death. We have shown that the balance between follicle-stimulating hormone and prolactin determines differentiation or apoptosis in 7th generation spermatogonia during newt spermatogenesis; however, the molecular mechanisms specifying their fate are poorly understood. Here we show that the newt RNA-binding protein (nRBP) plays a critical role in determining their fate. nRBP was identified as a clone whose mRNA is decreased by prolactin, resulting in the reduction of the protein, which is otherwise expressed predominantly in the spermatogonia. nRBP protein associated with the mRNA for newt programmed cell death protein 4 (nPdcd4) at the 3'-untranslated region. nRBP reduction increased nPdcd4 mRNA but decreased its protein. In a cell-free system, cytoplasmic extracts containing reduced amounts of nRBP and nPdcd4 protein induced apoptosis, whereas adding nRBP protein to the extracts blocked apoptosis. Furthermore, overexpression of nRBP protected cells from apoptosis, stabilized the chimeric transcript containing the nPdcd4 3'-untranslated region, and accelerated its translation. These data suggest that, in the absence of nRBP, nPdcd4 mRNA is not stabilized and its translation is suppressed, leading to apoptosis in the spermatogonia.

Identification of differentially expressed genes between osteoblasts and osteocytes

Osteocytes represent the most abundant cellular component of mammalian bones with important functions in bone mass maintenance and remodeling. To elucidate the differential gene expression between osteoblasts and osteocytes we completed a comprehensive analysis of their gene profiles. Selective identification of these two mature populations was achieved by utilization of visual markers of bone lineage cells. We have utilized dual GFP reporter mice in which osteocytes are expressing GFP (topaz) directed by the DMP1 promoter, while osteoblasts are identified by expression of GFP (cyan) driven by 2.3 kb of the Col1a1 promoter. Histological analysis of 7-day-old neonatal calvaria confirmed the expression pattern of DMP1GFP in osteocytes and Col2.3 in osteoblasts and osteocytes. To isolate distinct populations of cells we utilized fluorescent activated cell sorting (FACS). Cell suspensions were subjected to RNA extraction, in vitro transcription and labeling of cDNA and gene expression was analyzed using the Illumina WG-6v1 BeadChip. Following normalization of raw data from four biological replicates, 3444 genes were called present in all three sorted cell populations: GFP negative, Col2.3cyan + (osteoblasts), and DMP1topaz + (preosteocytes and osteocytes). We present the genes that showed in excess of a 2-fold change for gene expression between DMP1topaz + and Col2.3cyan + cells. The selected genes were classified and grouped according to their associated gene ontology terms. Genes clustered to osteogenesis and skeletal development such as Bmp4, Bmp8a, Dmp1, Enpp1, Phex and Ank were highly expressed in DMP1topaz + cells. Most of the genes encoding extracellular matrix components and secreted proteins had lower expression in DMP1topaz + cells, while most of the genes encoding plasma membrane proteins were increased. Interestingly a large number of genes associated with muscle development and function and with neuronal phenotype were increased in DMP1topaz + cells, indicating some new aspects of osteocyte biology. Although a large number of genes differentially expressed in DMP1topaz + and Col2.3cyan + cells in our study have already been assigned to bone development and physiology, for most of them we still lack any substantial data. Therefore, isolation of osteocyte and osteoblast cell populations and their subsequent microarray analysis allowed us to identify a number or genes and pathways with potential roles in regulation of bone mass.

Lin9 Is Required for Mitosis and Cell Survival during Early Zebrafish Development

LIN9 has been described as a regulator of G(1)/S and G(2)/M progression of the cell cycle in invertebrates and human cell lines. To elucidate the in vivo function of LIN9 during vertebrate development, we took advantage of the teleost zebrafish (Danio rerio). By means of antisense morpholinos we show here that Lin9-depleted embryonic cells accumulate in mitosis. Flow cytometry and confocal microscopy data demonstrate that the delay in mitotic progression is followed by apoptosis, which strongly manifests in the developing central nervous system. In accordance with these findings, we identified a cohort of Lin9-regulated genes required for different mitotic processes, including mitotic entry, metaphase/anaphase transition, and cytokinesis. Our data establish LIN9 as an essential regulator of mitosis in vertebrate development.

Automation of cDNA Synthesis and Labelling Improves Reproducibility

Background. Several technologies, such as in-depth sequencing and microarrays, enable large-scale interrogation of genomes and transcriptomes. In this study, we asses reproducibility and throughput by moving all laboratory procedures to a robotic workstation, capable of handling superparamagnetic beads. Here, we describe a fully automated procedure for cDNA synthesis and labelling for microarrays, where the purification steps prior to and after labelling are based on precipitation of DNA on carboxylic acid-coated paramagnetic beads. Results. The fully automated procedure allows for samples arrayed on a microtiter plate to be processed in parallel without manual intervention and ensuring high reproducibility. We compare our results to a manual sample preparation procedure and, in addition, use a comprehensive reference dataset to show that the protocol described performs better than similar manual procedures. Conclusions. We demonstrate, in an automated gene expression microarray experiment, a reduced variance between replicates, resulting in an increase in the statistical power to detect differentially expressed genes, thus allowing smaller differences between samples to be identified. This protocol can with minor modifications be used to create cDNA libraries for other applications such as in-depth analysis using next-generation sequencing technologies.