Intron-spanning Primers Research Articles

The pseudogene problem and RT-qPCR data normalization; SYMPK: a suitable reference gene for papillary thyroid carcinoma

In RT-qPCR, accuracy requires multiple levels of standardization, but results could be obfuscated by human errors and technical limitations. Data normalization against suitable reference genes is critical, yet their observed expression can be confounded by pseudogenes. Eight reference genes were selected based on literature review and analysis of papillary thyroid carcinoma (PTC) microarray data. RNA extraction and cDNA synthesis were followed by RT-qPCR amplification in triplicate with exon-junction or intron-spanning primers. Several statistical analyses were applied using Microsoft Excel, NormFinder, and BestKeeper. In normal tissues, the least correlation of variation (CqCV%) and the lowest maximum fold change (MFC) were respectively recorded for PYCR1 and SYMPK. In PTC tissues, SYMPK had the lowest CqCV% (5.16%) and MFC (1.17). According to NormFinder, the best reference combination was SYMPK and ACTB (stability value = 0.209). BestKeeper suggested SYMPK as the best reference in both normal (r = 0.969) and PTC tissues (r = 0.958). SYMPK is suggested as the best reference gene for overcoming the pseudogene problem in RT-qPCR data normalization, with a stability value of 0.319.

A genome-wide resource of intron spanning primers compatible for quantitative PCR and intron length polymorphism in rice

Whole genome sequence availability in rice has provided several advantages for genomics as well as other omics assisted applications. Genome-wide molecular markers are one of such availability that has exceptional importance in modern plant breeding. In the present study, a resource of intron-spanning primers (ISPs) was developed using whole genome sequence information of two rice subspecies, japonica (cv. Nipponbare) and indica (cv. 93-11). The ISPs were designed in a way that the PCR using a cDNA template will yield 60 to 100 base pair size amplicon ideal for the quantitative PCR analysis. Whereas, PCR using genomic DNA will amplify the introns, which are more prone to sequence variation. The sequence variation in the intron serves as an excellent marker resource. The application of ISPs was demonstrated by characterizing 12 diverse rice cultivars. A total of eight out of ten ISPs were found to be polymorphic. The resource will be helpful for the rice molecular biologist and breeder community.

A multiplex assay for the sensitive detection and quantification of male and female Plasmodium falciparum gametocytes

BackgroundThe transmission of malaria to mosquitoes depends on the presence of gametocytes that circulate in the peripheral blood of infected human hosts. Sensitive estimates of the densities of female gametocytes (FG) and male gametocytes (MG) may allow the prediction of infectivity to mosquitoes and thus a molecular estimate of the human infectious reservoir for transmission.MethodsA novel multiplex qRT-PCR assay with intron-spanning primers was developed for the parallel quantification of FG and MG. CCp4 (PF3D7_0903800) transcripts specific for FG and PfMGET (PF3D7_1469900) transcripts specific for MG were quantified in total nucleic acids. The assay was validated on sex-sorted gametocytes from culture material and on samples from clinical trials with gametocytocidal drugs. Synthetic RNA standards were generated for the two targets genes and calibrated against known gametocyte quantities.ResultsThe limit of detection was determined at 0.1 male and 0.1 female gametocyte/µL, which was equal to the limit of quantification (LOQ) for MG, while the LOQ for FG was 1 FG/µL. Results from previously reported clinical trials that used separate gametocyte qRT-PCR assays for FG (targeting Pfs25) and MG (targeting PfMGET) were reproduced with the multiplex assay. High levels of agreement between separate assays and the multiplex approach were observed (R2 = 0.9473, 95% CI 0.9314–0.9632, for FG measured by transcript levels of Pfs25 in qRT-PCR or CCp4 in multiplex; R2 = 0.8869, 95% CI 0.8541–0.9197, for MG measured by PfMGET in either single or multiplex qRT-PCR). FG and MG transcripts were detected in pure ring stage parasites at 10,000- and 100,000-fold reduced frequency for CCp4 and PfMGET, respectively. The CCp4 and PfMGET transcripts were equally stable under suboptimal storage conditions.ConclusionsGametocyte densities and their sex ratios can be determined in the presented one-step multiplex assay with higher throughput than single assays. The interpretation of low gametocyte densities at asexual parasite densities above 1000 parasites/µL requires caution to avoid false positive gametocyte signals from spurious transcript levels in ring stage parasites.

Human Epicardial Fat Expresses Glucagon-Like Peptide 1 and 2 Receptors Genes.

Epicardial adipose tissue (EAT) is an easily measurable visceral fat of the heart with unique anatomy, functionality, and transcriptome. EAT can serve as a therapeutic target for pharmaceutical agents targeting the fat. Glucagon-like peptide-1 (GLP-1) and GLP-2 analogues are newer drugs showing beneficial cardiovascular and metabolic effects. Whether EAT expresses GLP- 1 and 2 receptors (GLP-1R and GLP-2R) is unknown. RNA-seq analysis and quantitative real-time polymerase chain reaction (qRT-PCR) were performed to evaluate the presence of GLP-1R and GLP-2R in EAT and subcutaneous fat (SAT) obtained from 8 subjects with coronary artery disease and type 2 diabetes mellitus undergoing elective cardiac surgery. Immunofluorescence was also performed on EAT and SAT samples using Mab3f52 against GLP-1R. Our RNA-sequencing (RNA-seq) analysis showed that EAT expresses both GLP-1R and GLP-2R genes. qRT-PCR analysis confirmed that GLP-1R expression was low but detected by 2 different sets of intron-spanning primers. GLP-2R expression was detected in all patients and was found to be 5-fold higher than GLP-1R. The combination of accurately spliced reads from RNA-seq and successful amplification using intron-spanning primers indicates that both GLP-1R and GLP-2R are expressed in EAT. Immunofluorescence clearly showed that GLP-1R is present and more abundant in EAT than SAT. This is the first time that human EAT is found to express both GLP-1R and GLP-2R genes. Pharmacologically targeting EAT may induce beneficial cardiovascular and metabolic effects.

Abstract 442: Generation of an Abcg1 Knock Out Mouse on the Reversa Background

Background: Clinically HDL mediated reverse cholesterol transport (RCT) from macrophages has been shown to be inversely associated with carotid intima media thickness. Cholesterol efflux to mature HDL is mediated by ATP binding cassette transporter G1 (Abcg1). Abcg1 pays a key role in cholesterol transport with loss of function in macrophages and endothelial cells associated with significant cholesterol accumulation. However, mechanistic studies into the role of Abgc1 in plaque regression have been restricted due to the limitations of current regression models. Aims: To use TALENS mediated genomic editing to generate an Abcg1 knockout mouse on the REVERSA background to enable the investigation of its role in plaque regression. Methods and results: TALENs constructs were targeted to exon 3 upstream of the phosphate binding Walker A domain. TALEN mRNA was injected into REVERSA oocytes which were then implanted into foster mice. Founders were screened by Cel1 nuclease assay and sequencing. Three independent alleles were identified two of which create frameshift mutations (predicted to be null alleles) and one which resulted in a 3 amino acid deletion and a one amino acid substitution near the Walker A domain (potential hypomorphic allele). The two founder lines with frame shift mutations (KO 145 and 171) were taken forward for additional analysis. RNA extracted from primary macrophages from WT (REVERSA) and homozygous Abcg1 knockout mice was used to confirm the mutation was transcribed to RNA. Intron-spanning primers were designed and a product of the expected size was obtained and sequence analysis confirmed the insertion (KO-145) and deletion (KO-171) within the WALKER A domain of Abcg1. To ensure the mutations resulted in loss of function, a radioactive RCT assay was carried out in bone marrow derived primary macrophages. A significant decrease in RCT to HDL was observed in macrophages from both the KO-145 and KO-171 lines as expected there was no difference in RCT to ApoA1-I. Conclusions: Using genomic editing we have generated a gene specific knockout on the REVERSA background which will enable, for the first time, the investigation of the role of Abcg1 in plaque regression.

Evaluation of internal reference genes for quantitative expression analysis by real-time reverse transcription-PCR in somatic cells from goat milk

Reverse transcription (RT) quantitative real-time PCR (qPCR) is the most accurate and easy-to-perform technique to measure the expression level of a selected gene of interest by quantifying mRNA transcripts. The use of reference genes is commonly accepted as the most reliable approach to normalize RT-qPCR data and reduce possible errors generated in the quantification of gene expression. The optimal number and choice of reference genes are experimentally validated for specific tissues or cell types and experimental designs. To date, data on qPCR normalization in goats are scarce and the most suitable reference genes in this species have been identified for only a limited number of tissues. The aim of this study was to determine an optimal combination of stably expressed reference genes in caprine milk somatic cells (MSC) from healthy and infected mammary glands. For the purpose, we performed RT-qPCR for 10 commonly used reference genes from various functional classes and then determined their expression level in MSC from goats intramammary challenged with Staphylococcus aureus and in MSC from healthy controls, with a view to select genes whose stability would be unaffected under infection conditions. The geNorm and NormFinder algorithms were used for validating the reference genes. Furthermore, to demonstrate the importance of normalization of gene expression with appropriate reference genes, we tested the effect of using a combination of the least stable genes for expression analysis evaluation. On the basis of our evaluation, we recommend the use of a panel of reference genes that should include G6PD, YWHAZ, and ACTB for caprine MSC gene expression profiling. The expression of the 2 genes of interest, pentraxin-related protein (PTX3) and secreted phosphoprotein 1 (SPP1), was evaluated by RT-qPCR in all samples collected pre- and postinfection, and the recommended reference genes were used to normalize the data. Our study provides a validated panel of optimal reference genes for the identification of genes differentially expressed by qRT-PCR in caprine MSC. Moreover, we provided a set of intron-spanning primer sequences that could be suitable for gene expression experiments using SYBR Green chemistry on other caprine tissues and cells.

Upregulation of skeletal muscle inflammatory genes links inflammation with insulin resistance in women with the metabolic syndrome

The metabolic syndrome, a combination of interrelated metabolic risk factors, is associated with insulin resistance and promotes the development of cardiovascular diseases and type 2 diabetes mellitus. There is a close link between inflammation and metabolic disease, but the responsible mechanisms remain elusive. The aim of this study was to identify differentially expressed genes in insulin-resistant skeletal muscle tissue of women with the metabolic syndrome compared with healthy control women. Women with the metabolic syndrome (n = 19) and healthy control women (n = 20) were extensively phenotyped, insulin sensitivity was measured using a hyperinsulinaemic euglycaemic clamp, and a skeletal muscle biopsy was obtained. Gene expression levels were compared between the two groups by microarrays. The upregulated genes in skeletal muscle of the women with the metabolic syndrome were primarily enriched for inflammatory response-associated genes. The three most significantly upregulated of this group, interleukin 6 receptor (IL6R), histone deacetylase 9 (HDAC9) and CD97 molecule (CD97), were significantly correlated with insulin resistance. Taken together, these findings suggest an important role for a number of inflammatory-related genes in the development of skeletal muscle insulin resistance.

The NFKB1 Promoter Polymorphism (−94ins/delATTG) Alters Nuclear Translocation of NF-κB1 in Monocytes after Lipopolysaccharide Stimulation and Is Associated with Increased Mortality in Sepsis

Because the nuclear factor-κB (NF-κB) coupled pathway is believed to amplify inflammation prevailing in sepsis, the authors tested the hypotheses that the insertion-deletion polymorphism (-94ins/delATTG) (1) alters nuclear translocation of nuclear factor-κB and activator protein-1 (NF-κB1) in monocytes after lipopolysaccharide stimulation; (2) affects lipopolysaccharide-induced NF-κB1 messenger RNA expression, tumor necrosis factor α concentrations, and tissue factor activity; and (3) may be associated with increased 30-day mortality in patients with sepsis. Nuclear translocation of NF-κB1 in monocytes after lipopolysaccharide stimulation from healthy blood donors was performed with immunofluorescence staining (n = 5 each). Lipopolysaccharide-induced NF-κB1 messenger RNA expression was measured with real-time polymerase chain reaction (PCR; n = 60), tumor necrosis factor α concentrations with a multiplexing system kit (n = 60), and tissue factor activity with thromboelastometry (n = 105). In a prospective study, multivariate proportional hazard analysis tested 30-day mortality in patients with sepsis (n = 143). The homozygous deletion genotype compared with the homozygous insertion genotype was associated with a nearly twofold increase in nuclear translocation of NF-κB1 (P = 0.001), a threefold difference in NF-κB1 messenger RNA expression (P = 0.001), and a twofold increase in tissue factor expression (P = 0.021). The deletion allele in adults with severe sepsis was tested as an independent prognostic factor for 30-day mortality (hazard ratio, 2.3; 95% CI, 1.13-4.8; P = 0.022). Mortality was 25% for homozygous insertion genotypes but 41% for combined heterozygous deletion/homozygous deletion genotypes (P = 0.034). The deletion allele of the NFκB1 insertion-deletion (-94ins/delATTG) polymorphism is associated with increased 30-day mortality in patients with severe sepsis and increased reaction of the innate immune system.

201 IDENTIFICATION OF CANDIDATE X (INACTIVE)-SPECIFIC TRANSCRIPT (XIST) CODING REGION BY HOMOLOGY ANALYSIS OF SEQUENCE AND RELATIVE EXPRESSION ANALYSIS ON FERTILIZED EMBRYO AND PARTHENOTES IN PIG

X (inactive)-specific transcript (XIST) has been known as long noncoding RNA, which regulates X-chromosome inactivation in female mammals to provide dosage equivalence between male and females. Its important roles in early embryo and stem cells development have been suggested in humans and mice. However, its coding region has been unclear in pigs. To determine the coding region of XIST in pigs, we examined candidate XIST coding region using BLAST, PCR, and sequencing techniques. By comparing pig whole genome sequence (Sus scrofa 10.2) with human, murine, and bovine XIST transcript sequence using BLAST, we selected candidate coding regions of XIST in pig. The result showed that XIST is coded on the minus strand of NW_003612825 contig, and its length was nearly 32 kb, which was similar to the length of human and bovine XIST genes. In order to identify the coding region of XIST in this candidate sequence, we designed 24 primer pairs and RT-PCR was performed using porcine embryonic fibroblast cells (PEF) established from porcine male and female lines.The XIST transcripts were expressed in female PEF but not in male PEF. Thus, by designing candidate intron-spanning primers, we confirmed the existence of introns between the first and last exons. The amplicon sequence was analysed and 5 short exons (<400 bp) that have similarity to the XIST coding region of other characterised species were identified. Moreover, to examine the XIST coding strand in pig genome, we conducted strand-specific reverse transcription. With BLAST homology searches, we confirmed that XIST was coded on the negative strand of the contig on the X chromosome. To analyse stage and origin of XIST transcript in the pig, we subsequently performed real-time PCR using cDNA template synthesised from sperm, oocyte, 1, 2, 4, 8, morular, and blastocyst of fertilized embryo and parthenotes, and intron-spanning primer sets were used. The results revealed that XIST was expressed after maternal to zygote transition (4- to 8-cell stage in pig) and not expressed in mature germ cells (sperm and oocyte). Moreover, parthenotes showed a higher expression level compared with fertilized embryos after the 8-cell stage. These results verify the existence of XIST in pig early embryos and may contribute to the comparative analysis of XIST mechanisms in different species. Further study using FISH and methylation analysis may reveal its mechanisms of action. This work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (MEST; No. 2012006276) and the Biogreen 21 program (PJ0081382011) of the Rural Development Administration, Republic of Korea.

197 EXPRESSION OF MELATONIN-RELATED GENES IN RAT CUMULUS–OOCYTE COMPLEXES

Melatonin is a hormone usually associated with the modulation of circadian rhythms and the regulation of seasonal reproductive function. There is evidence that melatonin acts directly on the regulation of ovary function. The mRNA expression of melatonin membrane receptors genes was detected in mammalian and non-mammalian ovaries. In spite of melatonin receptors and Asmt (limiting enzyme in melatonin biosynthesis) genes being present in cattle cumulus–oocyte complexes (COC), no information regarding rat COC has been reported. The aim of this study was to investigate the expression of melatonin receptor (Mt2) and melatonin synthesis enzyme (Asmt) genes at different meiotic cell cycle stages in COC from 27-day-old female rats. All the procedures involving animals were approved by the Animal Care Committee of the Institute of Biomedical Sciences. To obtain germinal vesicle (GV) immature COC from ovarian follicles, rats were treated with 20 IU equine chorionic gonadotropin (eCG) for induction of follicular development and killed by euthanasia 48 h later. To obtain COC at MII oocyte stage from oviducts, rats were injected with 20 IU of eCG and 48 h later with 20 IU of human chorionic gonadotropin (hCG), and then killed after 14 to 16 h. The oocytes in COC were separated from their cumulus cells by repeated pipetting through a narrow-bore pipette in culture medium. Oocytes and cumulus cells total RNA were isolated using a Trizol reagent (Invitrogen Corp., Carlsbad, CA, USA). Pools of 80 COC per cDNA sample were used. Quantitative real-time PCR (qRT-PCR) was performed (7500 Real-Time PCR System; Applied Biosystems Inc., Foster City, CA, USA) with 25-µL reactions containing 2 µL of cDNA (10 ng µL–1), SYBR green (Power SYBR Green; Applied Biosystems Inc.), and 400 nM specific intron-spanning primers. A set of 10-fold serial dilutions of each internal standard (102 to 106 copies/2 µL) was used to generate a standard curve. Transcript numbers were determined by the system software and normalized using the geometric mean calculated from the reference genes: Actb and Rpl37a. All the results were plotted as the mean ± SEM, and 4 replicates were performed. Unpaired t-test was used to evaluate the cell type (oocyte v. cumulus cells) differences. The qRT-PCR analyses revealed the presence of transcripts of the Mt2 gene only in oocytes from immature (GV) and mature (MII) COC. At both maturational stages, the copy numbers for Asmt in oocytes were significantly higher than in cumulus cells. The results confirm the presence of the Asmt gene in rat COC and suggest the possible involvement of these cells on melatonin biosynthesis. The presence of Mt2 transcript in immature and mature oocytes also suggests the potentially important role of melatonin in regulating the rat meiotic cellular cycle. Research supported by FAPESP.

Reprogramming and Characterization of Cord Blood Derived Stem Cells by Synthetic mRNAs: Potential for Cord Blood Stem Cell Regenerative Therapy

AbstractAbstract 4748 Background:Recent development of a synthetic mRNA-based technology for reprogramming somatic cells to pluripotency has overcome the challenge faced by DNA-based reprogramming methods (Warren L et al Cell stem cell 2010). This method involves a daily transfection of a five-factor cocktail including modified mRNAs encoding Klf4, cMyc, Oct4, Sox2, and Lin28 (KMOSL) for 17 days. Cord blood is a rich source for stem cells that could be novel sources for reprogramming (Liao/Cairo et al Exp Hem 2010). An important source of cord blood stem cells include unrestricted somatic stem cells (USSCs), which have been demonstrated to have multi-lineage differentiation abilities (Kögler G et al J. Exp Med 2004; Liao/Cairo, ASBMT 2010). Reprogramming cord blood derived USSCs have significant translational applications for a number of pediatric diseases. Goal:To determine the reprogramming efficiency of cord blood derived USSCs utilizing synthetic mRNAs. Method:USSCs were isolated from HUCB in the presence of 30% FBS and 10−7M dexamethasone, and characterized for their stem cell properties. USSCs were further sorted based on the expression of surface marker SSEA4 and reprogrammed using Stemgent mRNA reprogramming system, following daily transfection of KMOSL mRNA cocktail for 4 hours. At the end of each transfection, the culture was replaced by fresh Pluriton Medium supplemented with B18R protein. Human fibroblasts were reprogrammed in parallel as a control. Upon the appearance of iPS-like colonies, DyLight 488 TRA-1–60 antibody was added to the culture to identify iPS cells. The positively stained colonies were then manually picked and expanded in standard ES medium. Expression of ES factors and the formation of teratoma in vivo were used to confirm pluripotency of the derived cells. Results:USSCs share typical MSC cell surface markers and can be distinguished from MSCs based on their expression of d-like 1/preadipocyte factor 1 (DLK-1). 57.46% of USSCs (± 10.97%) displayed stage-specific embryonic antigen-4 (SSEA-4); however USSCs were negative for TRA-1–60. Approximately 33% (± 4.8%) of the USSCs were also stained positive for alkaline phosphatase activity. Moreover, compared to a complete absence of Oct4 and Nanog gene transcription in human fibroblasts, a minimal level of such pluripotency gene expression was detected in USSCs using isoform-specific and intron-spanning primers. Treatment of USSCs with 5-azaCytidine further led to a 10-fold increase in the expression of the Oct4 and Nanog genes in USSCs, but not in fibroblasts. Consistently, the upstream regulatory regions of both Oct4 and Nanog genes exhibited an intermediate level of DNA methylation in USSCs as compared to human ES and fibroblasts. These data suggest the plasticity of USSCs in reprogramming. SSEA4-positive USSCs were then used for daily transfection with mRNA KMOSL cocktail. The iPS-like colonies started to appear in the USSC reprogramming plates on day 11. TRA-1–60 live staining demonstrated that most of the colonies was stained positive for TRA-1–60 and the efficiency of iPS derivation from USSCs was about 0.2%. In comparison, the iPS-like colonies were observed in the fibroblast reprogramming plates on day 15–16 of transfection and the derivation efficiency was five-fold lower than that of USSCs. This demonstrated that USSCs can be reprogramming more efficiently than fibroblasts. The TRA-1–60 positive colonies were manually picked from the USSC reprogramming plates and expanded in ES medium. The colonies were also stained positive for Oct4, Nanog, Sox2 and TRA1–81. The in vivo teratoma assay is now under investigation. We are also investigating the reprogramming of USSCs using a single mRNA factor, in combination with small molecules, including PD0325901 and SB431542. Disclosures:No relevant conflicts of interest to declare.

Evaluation of Internal Reference Genes for Quantitative Expression Analysis by Real-Time PCR in Ovine Whole Blood

The use of reference genes is commonly accepted as the most reliable approach to normalize qRT-PCR and to reduce possible errors in the quantification of gene expression. The most suitable reference genes in sheep have been identified for a restricted range of tissues, but no specific data on whole blood are available. The aim of this study was to identify a set of reference genes for normalizing qRT-PCR from ovine whole blood. We designed 11 PCR assays for commonly employed reference genes belonging to various functional classes and then determined their expression stability in whole blood samples from control and disease-stressed sheep. SDHA and YWHAZ were considered the most suitable internal controls as they were stably expressed regardless of disease status according to both geNorm and NormFinder software; furthermore, geNorm indicated SDHA/HPRT, YWHAZ/GAPDH and SDHA/YWHAZ as the best reference gene combinations in control, disease-stressed and combined sheep groups, respectively. Our study provides a validated panel of optimal control genes which may be useful for the identification of genes differentially expressed by qRT-PCR in a readily accessible tissue, with potential for discovering new physiological and disease markers and as a tool to improve production traits (e.g., by identifying expression Quantitative Trait Loci). An additional outcome of the study is a set of intron-spanning primer sequences suitable for gene expression experiments employing SYBR Green chemistry on other ovine tissues and cells.

Purinergic signalling mobilizes mitochondrial Ca2+ in mouse Sertoli cells

Intimate bidirectional communication between Sertoli cells and developing germ cells ensures the integrity and efficiency of spermatogenesis. Yet, a conceptual mechanistic understanding of the physiological principles that underlie Sertoli cell autocrine and paracrine signalling is lacking. Here, we characterize a purinergic Ca(2+) signalling network in immature mouse Sertoli cells that consists of both P2X2 and P2Y2 purinoceptor subtypes, the endoplasmic reticulum and, notably, mitochondria. By combining a transgenic mouse model with a dedicated bioluminescence imaging device, we describe a novel method to monitor mitochondrial Ca(2+) mobilization in Sertoli cells at subcellular spatial and millisecond temporal resolution. Our data identify mitochondria as essential components of the Sertoli cell signalling 'toolkit' that control the shape of purinergic Ca(2+) responses, and probably several other paracrine Ca(2+)-dependent signals.

Mosaic Pattern of Cpg Island methylation in Unrestricted Somatic Cells (USSC) Derived From Human Umbilical Cord Blood (HUCB).

AbstractAbstract 2608 Introduction:HUCB has been described to contain hematopoietic multi-lineage progenitor cells that contribute to the success in treating malignant and non-malignant diseases (Cairo et al, Blood, 1997). We demonstrated that multi-lineage progenitor cells derived from HUCB can differentiate into cells representative of the 3 germ layers in vitro (Vandeven/Cairo et al, Exp Hem, 2007). Zaehres et al (Exp Hem, 2010) have described another population of primitive stem cells, USSCs, as a new potential source to generate iPS cells following retroviral transduction. This suggests that USSCs are a strong candidate as a source for developing patient specific or HLA matched iPSC banks. Since using retroviral iPS cells are challenged with the possibility of oncogene reactivation; gene integration free methods of generating iPS cells such as small molecule treatment to activate ES transcription factor genes are needed. Furthermore, the epigenetic modification of the USSCs at the key ES transcription factors has not been described and this information will provide insights on the differentiation potential of USSCs and their capacity to reprogramming. Objective:To determine the DNA methylation patterns in the core regulatory regions, including both enhancer and promoter of ES transcription factors Oct4 and Nanog in USSCs prior to and following gene transcription effects of DNMT1 inhibition by treatment with 5-azacytidine. Methods:USSCs were derived from HUCB mononuclear cells in 30% FBS and 10-7M dexamethasone (Kogler, J Exp Med, 2004). The USSC population was confirmed by flow cytometry and their fibroblastic morphology. The cells were passaged in the same medium without dexamethasone. Bisulfite sequencing was performed to characterize the CpG island methylation in the regulatory regions of Oct4 (from 2563 bp upstream to 250 bp downstream of Oct4 transcription start site) and Nanog (from 1449 bp to 82 bp upstream of the Nanog transcription start site). RT-PCR and qPCR were conducted to determine the expression levels of Nanog, Oct4 and Sox2, using isoform specific and intron spanning primers. 3mM 5-azacytidine was used to treat USSCs for demethylation studies and the RNA was collected 24 hours following treatment. The results were compared to human embryonic stem cells and human foreskin fibroblasts as positive and negative controls, respectively. Results:USSCs were derived from 50% (n=25) of HUCB; with 1–10 colonies from each successfully derived cord blood. Flow cytometry indicated that USSCs were lineage negative and express overlapping but distinct cell surface markers with MSCs; positive for CD73, CD90, CD146, CD50, but negative for CD106. Bisulfite sequencing of USSCs demonstrated a mosaic methylation pattern of CpG islands at the regulatory sites of both Oct4 and Nanog. An average of 65% and 47% of the CpGs were unmethylated in the enhancer and promoter regions of Nanog, respectively. 56% were unmethylated at the enhancer of Oct4 while the promoter was heavily methylated, except for the 400 bp region that spans the Oct4 transcription start site, which was 80% unmethylated. This is consistent with the RT-PCR results showing a low but consistent level of Nanog, Oct4 and Sox2 (Figure 1). Based on q-PCR using isoform-specific and intro-spanning primers, we determined that USSCs have about 20-and 400- fold higher levels of Nanog and Oct4 expression, respectively,compared to fibroblasts. Following a 24hr exposure to a DNMT1 inhibitor, 5-azacytidine, to the USSCs, there was a 10-fold increase in the mRNA expression of Oct4, Nanog, and Sox2. [Display omitted] Conclusions:HUCB derived USSCs have a mosaic pattern of CpG island methylations in the distal and proximal regions of key ES transcription factors, Oct4 and Nanog. This is supported by a consistent low expression level of these genes. The mosaic pattern of CpG islands seems to be more malleable by small molecule perturbation; 3mM of 5-azacytidine appeared to significantly increase the Oct4 and Nanog expression. We hypothesize that due to their semi-permissive chromatin structure at the core regulatory regions in key ES transcription factors, HUCB derived USSCs are likely to be a more optimal choice of small molecule derived induced pluripotent stem cells compared to other cell types. Disclosures:No relevant conflicts of interest to declare.

Leptin mRNA in bovine spermatozoa

Leptin is a hormonal product of the ob gene, which is involved in energy metabolism and reproduction. Expression of leptin and its receptor in reproductive organs suggests that in addition to its endocrine effects, it may have paracrine/autocrine effects on reproduction. Also, the expression of leptin in human and pig spermatozoa and its secretion by these cells suggest a direct role for leptin in sperm physiology. The present study was the first to investigate the presence of the leptin mRNA transcript in Holstein cattle spermatozoa by reverse transcriptase-polymerase chain reaction (RT-PCR) analysis. Total RNA was extracted from different concentrations of ejaculated or epididymal sperm cells and after synthesis of complementary DNA was subjected to PCR amplification. In both ejaculated and epididymal spermatozoa, leptin transcript was amplified with first set of primers. The first amplicon was used for a nested PCR with second set of primers. Intron-spanning primers were located in exons 2 and 3 to rule out the possibility of a DNA contamination. Amplificons of leptin were only detectable when more than 100 million epididymal or ejaculated spermatozoa was used. Presence of leptin mRNA in the bull spermatozoa suggests that leptin might be involved in the physiological processes of bovine spermatozoa, which remain to be further clarified.

Increased gene expression of diacylglycerol kinase eta in bipolar disorder

Bipolar disorder (BD) is a highly heritable neuropsychiatric illness characterized by recurrent episodes of depression and mania or hypomania. Recent genome-wide association studies of BD have proposed novel candidates, including the gene encoding the η isoform of diacylglycerol kinase (DGKH; Baum et al. 2008a). The association of this gene with BD was replicated at the haplotype level in a Sardinian sample (Squassina et al. 2009), but not for individual SNPs or as a predictor of lithium response (Manchia et al. 2009). Diacylglycerol kinase (DGK) regulates the intracellular concentration of diacylglycerol, which is a main component of inositol signalling systems targeted by the major anti-BD agent, lithium (reviewed in van Blitterswijk et al. 2000; see also Harwood, 2005, for discussion of the inositol-depletion hypothesis and lithium and BD). DGKH is highly expressed in brain and is also glucocorticoid-inducible and stress-responsive (Klauck et al. 1996; Murakami et al. 2003). Here, we present evidence that gene expression of DGKH is increased in BD. We quantified DGKH mRNA by real-time quantitative polymerase chain reaction in a collection of 100 post-mortem brain-tissue samples donated by The Stanley Medical Research Institute. All samples were derived from the dorsolateral prefrontal cortex and originated from three diagnostic groups: BD (n = 31), schizophrenia (n = 35) and unaffected controls (n = 34). We used intron-spanning primers TGA CAG CAC AGA AAC AGA TGA AT and GGA GAC CGA GGT GCA GTT T as well as a fluorescent probe (Universal Probe Library no. 69, Roche Applied Science, USA) under amplification conditions described previously (Wendland et al. 2009). Experiments were done after the specimen code was broken and they were thus unblinded. Using G*Power version 3.1.2 (Faul et al. 2007), we determined our sample had some power (56%) to detect medium effect sizes (f = 0.25) and excellent power (94%) for large effect sizes (f = 0.4) in a fixed-effects omnibus analysis of variance test. The entire dataset has been uploaded to The Stanley Medical Research Institute databank (http://www.stanleyresearch.org/brain). We did not apply multiple testing correction for all comparable expression genetics studies uploaded to this databank. All experiments were conducted under protocols approved by the Institutional Review Board of the National Institute of Mental Health Division of Intramural Research Programs in Bethesda, MD. One-way analysis of variance showed that the three diagnostic groups differed significantly in their DGKH mRNA expression levels (F2,97 = 4.44, p = 0.01). As shown in Fig. 1, BD samples displayed significantly increased DGKH levels (Tukey’s post-hoc analysis, p <0.05), with the mean expression level being about 25% higher in BD than in controls. We also quantified two known DGKH transcript variants (Murakami et al. 2003) with transcript-specific probes but did not observe significant differences; moreover, we did not observe significant sex or age effects (data not shown). Fig. 1 Expression levels of DGKH mRNA in post-mortem brain tissue samples of three diagnostic groups. A total of 100 samples [bipolar disorder (BD, n = 31), schizophrenia (SZ, n = 35) and unaffected controls (n = 34)] were analysed by quantitative polymerase ... We genotyped all samples for two SNPs within the genomic DGKH locus, rs1170191 and rs1012053, that remained associated with BD in a meta-analysis (Baum et al. 2008b). Allelic and genotypic frequencies did not significantly differ between the three diagnostic groups for both SNPs. There was no association of either SNP with DGKH mRNA levels, even after inclusion of diagnosis as covariate (data not shown). Our data suggest that increased gene expression of DGKH is involved in the pathogenesis of BD. Interestingly, the BD disorder group did not differ significantly from the schizophrenia group (although the latter was also not significantly different from normal controls, Fig. 1), raising the possibility that the observed increase is not specific for BD. The average expression level was 20%higher in schizophrenia donors compared to controls, but this did not reach statistical significance in the post-hoc test. Larger post-mortem and other functional studies with greater power might clarify the role of DGKH in other psychiatric disorders. The DGKH over-expression hypothesis described above can be tested in future genetics studies once expression quantitative trait loci have been identified for DGKH. Further, model organisms over-expressing Dgkh may allow further insight into pathogenetic mechanisms of BD. The lack of correlation of SNPs that first implicated DGKH in BD with expression levels suggests that other variants conferring allele-specific expression may result in stronger association signals with the disorder. It thus seems warranted to identify variants correlating with high levels of DGKH mRNA or gain-of-function coding polymorphisms as carriers of these alleles can be postulated to be at increased risk for BD. It is our hope that this study might contribute to the elucidation of the pathogenesis of this debilitating disorder.

Genomics in cereals: from genome-wide conserved orthologous set (COS) sequences to candidate genes for trait dissection

Recent updates in comparative genomics among cereals have provided the opportunity to identify conserved orthologous set (COS) DNA sequences for cross-genome map-based cloning of candidate genes underpinning quantitative traits. New tools are described that are applicable to any cereal genome of interest, namely, alignment criterion for orthologous couples identification, as well as the Intron Spanning Marker software to automatically select intron-spanning primer pairs. In order to test the software, it was applied to the bread wheat genome, and 695 COS markers were assigned to 1,535 wheat loci (on average one marker/2.6 cM) based on 827 robust rice-wheat orthologs. Furthermore, 31 of the 695 COS markers were selected to fine map a pentosan viscosity quantitative trait loci (QTL) on wheat chromosome 7A. Among the 31 COS markers, 14 (45%) were polymorphic between the parental lines and 12 were mapped within the QTL confidence interval with one marker every 0.6 cM defining candidate genes among the rice orthologous region.

Expression of multiple catechol‐o‐methyltransferase (COMT) mRNA variants in human brain

Catechol-o-methyltransferase (COMT) is important for modulating dopamine levels, prefrontal cortex (PFC) function, and several psychiatric phenotypes. A single COMT mRNA has been described in human brain, which gives rise to membrane-bound (MB)- and soluble (S)-COMT proteins. In addition, we have recently described a novel COMT protein isoform in the human PFC, suggesting that there are more COMT gene products expressed than are currently appreciated. Therefore, we have investigated whether variant COMT mRNAs are present in human brain. We used reverse transcription-PCR (RT-PCR) to screen systematically for variant COMT mRNAs in human frontal cortex. Intron-spanning primers were used for exon-to-exon PCR reactions; additionally, specific primers were designed to sequences in the NCBI Aceview database. The identity of amplicons was confirmed by sequencing, and their regional distributions and 3' untranslated regions (UTRs) were characterised using RT-PCR. We detected 7 COMT variant mRNAs, resulting from both insertions and deletions within the known COMT brain transcript. Several of the variants alter the predicted coding sequence. Three of these variants correspond to sequences within the Aceview database and could be reliably amplified, while the remaining four do not correspond to any expressed sequence tags and were amplified only once. The regional distributions of these transcripts are described. The results demonstrate multiple COMT mRNAs in human brain, revealing an additional complexity to the biology of COMT. The alternate gene products may be of significant functional importance, and differentially impacted by polymorphisms within the COMT gene.

PD6-1-2: ERCC1 and TOPOIIa mRNA levels predict response to platinum/etoposide chemotherapy and survival of small-cell lung cancer patients.

ERCC1 and TOPOIIa mRNA levels predict response to platinum/ etoposide chemotherapy and survival of small-cell lung cancer patients. Selvaggi, Giovanni1 Ceppi, Paolo2 Giai Levra, Matteo1 Novello, Silvia1 Longo, Marina1 Cappia, Susanna2 Papotti, Mauro2 Volante, Marco2 Scagliotti, Giorgio V.1 1 Thoracic Oncology at Ospedale San Luigi, University of Torino, Orbassano, Italy 2 Pathology at Ospedale San Luigi, University of Torino, Orbassano, Italy Background: This study was designed to evaluate the predictive role of mRNA quantification of three genes potentially related to platinum/ etoposide therapy in small-cell lung cancer (SCLC): ERCC1, RRM1 and TOPOIIα. Methods: A retrospective analysis was therefore performed on 103 consecutive patients with either limited stage (LS) or extensive stage (ES) SCLC. Total RNA was extracted from microdissected sections of 103 formalin-fixed, paraffin embedded pre-treatment biopsies of SCLC patients. Relative quantification for ERCC1, RRM1 and TOPOIIα plus an internal reference gene (b-actin) was done by Real Time PCR using intron-spanning primers. ERCC1 was evaluated also by immunohistochemistry (IHC) with a semi-quantitative score. Results: Eighty-five samples (83%) were successfully amplified by Real Time PCR and a gene expression level was detectable in all samples. Out of these 85 cases, 45 (53%) had LS SCLC and 40 (47%) ES SCLC; overall response rate (RR) to chemotherapy was 65% (56 cases), median survival was 9.9 months. IHC staining of ERCC1 was positive in 2 out of 85 patients with no correlation with clinico-pathological factors. A significant correlation emerged between ERCC1 and RRM1 mRNA levels (Rs=0.34, p=0.001). Low TOPOIIα expression was associated to increased RR in LS patients (p=0.025) and TOPOIIα levels were lower in patients who had a complete response than in patients with either partial responses or no response (p=0.01). At univariate analysis LS patients with low ERCC1 mRNA levels had longer median survival (14.9 months vs. 9.9 months, p=0.01), while RRM1 and TOPOIIα levels showed no significant influence on survival. At multivariate analysis, ERCC1 was confirmed as an independent prognostic factors for survival in LS patients, together with performance status (PS) and response to chemotherapy. No significant prognostic role was found for ERCC1, RRM1 and TOPOIIα in patients with ES SCLC. Conclusions: Pharmacogenomics have been widely studied in advanced NSCLC, while poor efforts have so far been devoted on such topic in SCLC. ERCC1 and TOPOIIα mRNA levels could represent new reliable markers to predict response to treatment and clinical outcome in subgroups of SCLC patients. The path to the individualization of therapy through selection of patients based on pharmacogenomic data should be pursued to improve prognosis of SCLC. PD6-1-3 SCLC, Mon, 16:00 17:30

EXPRESSION OF PERIATTACHMENT FACTOR mRNA DURING OVINE CONCEPTUS DEVELOPMENT

Periattachment factor (PF) mRNA was first identified in the bovine conceptus at markedly greater concentrations on d 17.5 than d 15.5 of gestation. Since that time, work in our lab has identified PF mRNA in trophoblast-derived tissues, but not fetal tissues, of the early ovine and equine conceptus, as well as the human placenta, and determined that it is a nuclear protein. Complementary DNA (cDNA) homologues have also been identified in colon and mammary carcinomas, as well as the kidney and intestine. The purpose of this study was to characterize expression of PF mRNA in the developing ovine conceptus, and to compare this to a gene of known importance during ruminant conceptus development, interferon-tau (INFt). Conceptuses were collected from mature ewes on d 11, 13, 15, 16, 17, 21, and 30 post-mating (n = 4 to 6/day). Total cellular RNA was isolated from individual conceptuses and the cDNA generated was quantified by spectrophotometry. Intron-spanning primers were developed for ovine PF (oPF), and primer and probe sequences for ovine INFt (oINFt) were donated. Both genes were normalized to ovine glyceraldehyde-3-phosphate dehydrogenase (oGAPDH) after determining that oGAPDH concentrations did not differ among time points. For quantitative RT-PCR (qRT-PCR) analysis, a standard curve was created from 1 × 102 to 1 × 10−6 picograms. Equal amounts of cDNA for each sample were subjected to 40 cycles of qRT-PCR. GAPDH normalized oPF and oINFt mRNA concentrations were subjected to polynomial regression analysis, and comparisons between individual days were made by Tukey's-HSD. oPF exhibited a quadratic expression pattern (P < 0.02). No oPF mRNA was detected in any d 11 conceptus sample. From d 13 oPF increased to a peak at d 16 before declining. oPF expression was significantly greater on d 16 compared to d 30. oINFt mRNA exhibited a linear decrease (P < 0.01), from highest expression at d 13 to low amounts at d 30. oINFt expression on d 13 and 15 was greater than on d 21 and 30 (P<0.05). The expression pattern of oINFt mRNA will be used in subsequent oPF knockdown experiments as a marker of normal conceptus development. Peak expression of oPF mRNA in the conceptus occurs during the time of rapid elongation and initial attachment of the conceptus to the uterine endometrium. Furthermore, there is considerable embryonic loss during this time. While the exact function of oPF is unknown, the robust increase in mRNA during a critical time point in early trophoblast expansion, along with its identification in other expanding/regenerating epithelium, may suggest an important role in conceptus development. This project was supported by National Research Initiative Competitive Grant no. 2005-35203-15885 from the USDA Cooperative State Research, Education, and Extension Service. (poster)