Housekeeping Genes For Normalization Research Articles

The effects of the Rheum ribes plant extract on inflammation, extracellular matrix remodeling, and obesity suggest a therapeutic potential

The prevalence of obesity is increasing in the world, and the Type II diabetes associated with obesity led researchers to seek alternative methods to treat these two chronic diseases. In the case of obesity and diabetes, changes occur in the levels of inflammatory mediators. A study was conducted to investigate the molecular mechanism of the Rheum ribes L. plant regarding obesity and inflammation. Differentiated 3T3-L1 mouse cell lines were used as an experimental model. A dose-response relationship was established to determine at what dose and time of treatment the R. ribes L. plant extract would act effectively. To assess expression on the transcriptional level, q-PCR analyses were performed. The primers to evaluate the expression levels of genes such as Dgat1, Lpl, Fasn, ColV, Il-6, and Mcp1, which are known to be associated with obesity and insulin resistance, inflammation, and cell skeletal restructuring was designed using NCBI sequences. 18S was chosen as the housekeeping gene for normalization. It was found that applying 50µg/mL and 100µg/mL of R. ribes root extract to 3T3-L1 adipocyte cells for 24 and 48h resulted in anti- obesity and anti-inflammatory effects on the genes examined at the transcriptional level. It is an effective study to understand the molecular mechanisms by which R. ribes, which is known to have anti-diabetic, anti-obesity and anti- inflammatory activities, and to establish a link between these activities.

OR19-01 IHC-Guided Capture of Adrenocortical Zones from Archival Formalin-Fixed, Paraffin-Embedded Adrenal Tissue Allows the Study Of RNA Expression Profiles

Introduction: Characterization of human adrenal RNA has relied on fresh tissue, or laser capture from OCT-embedded frozen tissue. These approaches have allowed identification of differences between zones of the adrenal cortex, but the need for fresh or frozen material has limited access for most researchers and has limited the number of samples that can be studied. In order to expand tissue availability and researcher access, we examined the use of archival formalin-fixed, paraffin-embedded (FFPE) adrenals for the capture and analysis of RNA from each adrenal zone.Methods: 13 normal adrenals were obtained from deceased renal donors. Following FFPE, tissue serial sections were examined for zonal histology. Immunohistochemistry (IHC) was used to guide the capture of specific adrenal zones from FFPE tissue. CYP11B2, CYP17A1, and CYB5A IHC was used to visualize the zona glomerulosa (ZG), zona fasciculata (ZF), and zona reticularis (ZR) respectively. Serial unstained sections were then dissected under a dissection microscope based on IHC-guided visualization of the ZG, ZF, and ZR. Following capture of zones and isolation of RNA, quantitative PCR (qPCR) was used to examine zonal expression of transcripts for the IHC markers. Using housekeeping genes for normalization, specific zonal markers identified successfully isolated zones.Results: Adrenals with clear zonal expression of CYP11B2 (ZG), HSD3B2 (ZG/ZF), and CYB5A (ZR) were included for study. Based on marker inclusion criteria, 3 ZG, 3 ZF, 3 ZR, and 3 medulla samples were studied. Using these samples, confirmatory markers of ZG (VSNL1), ZF (CYP17A1), ZR (SLC27A2), and medulla (TH) were analyzed by qPCR. Levels of these transcripts segregated as expected in the adrenal zones.Discussion: The use of archival normal and pathologic adrenal tissue for RNA analysis would increase the number of samples available to many research groups. Our data demonstrates the ability to capture and use FFPE tissue for the capture and analysis of RNA. Future comparison of normal adrenal zones to adrenal tumor phenotypes will improve our understanding of tumor biology and provide potential genetic markers for targeted therapeutics.

Real-time PCR quantification of spliced X-box binding protein 1 (XBP1) using a universal primer method.

X-box binding protein 1 (XBP1) mRNA processing plays a crucial role in the unfolded protein response (UPR), which is activated in response to endoplasmic reticulum (ER) stress. Upon accumulation of the UPR-converted XBP1 mRNA splicing from an unspliced (u) XBP1 (inactive) isoform to the spliced (s) XBP1 (active) isoform, inositol-requiring enzyme 1 α (IRE1α) removes a 26-nucleotide intron from uXBP1 mRNA. Recent studies have reported the assessment of ER stress by examining the ratio of sXBP1 to uXBP1 mRNA (s/uXBP1 ratio) via densitometric analysis of PCR bands relative to increased levels of sXBP1 to uXBP1 using a housekeeping gene for normalization. However, this measurement is visualized by gel electrophoresis, making it very difficult to quantify differences between the two XBP1 bands and complicating data interpretation. Moreover, most commonly used housekeeping genes display an unacceptably high variable expression pattern of the s/uXBP1 ratio under different experimental conditions, such as various phases of development and different cell types, limiting their use as internal controls. For a more quantitative determination of XBP1 splicing activity, we measured the expression levels of total XBP1 (tXBP1: common region of s/uXBP1) and sXBP1 via real-time PCR using specific primer sets. We also designed universal real-time PCR primer sets capable of amplifying a portion of each u/s/tXBP1 mRNA that is highly conserved in eukaryotes, including humans, monkeys, cows, pigs, and mice. Therefore, we provide a more convenient and easily approachable quantitative real-time PCR method that can be used in various research fields to assess ER stress.

Transcriptome profiling of coriander: a dual purpose crop unravels stem gall resistance genes

Stem gall (Protomyces macrosporus Unger), a serious disease that affects leaves, petioles, stems and fruits of coriander (Coriandrum sativum L.) causing heavy loss in yield. Genetic improvement of coriander for stem gall disease is indispensable. Coriander cultivars of stem gall resistance (ACr-1) and susceptible (CS-6) leaf samples were utilized and transcriptome sequenced using Illumina NextSeq500 platform. After trimming low-quality reads and adapter sequences, a total of 49,163,108 and 43,746,120 high-quality reads were retained and further assembly resulted validated transcripts of 59,933 and 56,861. We have predicted 52,506 and 48,858 coding sequences (CDS) ofwhich 50,506 and 46,945 were annotated using NCBInr database. Gene ontology analysis annotated 19,099 and 17,625 terms; pathway analysis obtained 24 different functional pathway categories; signal transduction, transport, catabolism, translation and carbohydrate metabolism pathways etc. were dominated. Differentially expressed genes analysis predicted 13,123 CDS commonly expressed of which 431 and 400 genes were significantly upregulated and downregulated, respectively, in which Rgenes, stress inducible transcription factors such as ERF, NAC, bZIP, MYB, DREB and WRKY and antifungal related genes were predicted. The real-time PCR analysis of HSP20 gene expression in resistance showed upregulation by 10-fold over susceptible sample and 18s used as a housekeeping gene for normalization. The present results provide an insights into various aspects underlying the development of resistance to stem gall in coriander.

Contribution of molecular analysis to the typification of the non-functioning pituitary adenomas.

AimThe WHO Classification of Tumours of Endocrine Organs considers the inmunohistochemical characterization of pituitary adenomas (PA) as mandatory for patient diagnosis. Recent advances in the knowledge of the molecular patterns of these tumours could complement this classification with gene expression profiling.MethodsWithin the context of the Spanish Molecular Registry of Pituitary Adenomas (REMAH), a multicentre clinical-basic research project, we analysed the molecular phenotype of 142 PAs with complete IHC and clinical information. Gene expression levels of all pituitary hormones, type 1 corticotrophin-releasing hormone receptor, dopamine receptors and arginine vasopressin receptor 1b were measured by quantitative real-time polymerase chain reaction. In addition, we used three housekeeping genes for normalization and a pool of nine healthy pituitary glands from autopsies as calibration reference standard.ResultsBased on the clinically functioning PA (FPA: somatotroph, corticotroph, thyrotroph and lactotroph adenomas), we established the interquartile range of relative expression for all genes studied in each PA subtype. That allowed molecularly the different PA subtypes, including the clinically non-functioning PA (NFPA). Afterwards, we estimated the concordance of the molecular and immunohistochemical classification with clinical diagnosis in FPA and between them in NFPA. The kappa values were higher in molecular than in immunohistochemical classification in FPA and showed a bad concordance in all NFPA subtypes.ConclusionsAccording to these results, the molecular characterization of the PA complements the IHC analysis, allowing a better typification of the NFPA.

Normalization of a NAFLD gene expression signature

We read with great interest the article by Moylan et al.1 in which the authors demonstrate that a 64-gene profile reproducibly differentiated severe nonalcoholic fatty liver disease (NAFLD) from mild NAFLD, and that a 20-gene subset within this profile correlated with NAFLD severity, independent of other known factors impinging on NAFLD progression. The need for accurate risk stratification for NAFLD is urgent because the disease is epidemic, imposing a public health burden, and cirrhosis and/or hepatocellular carcinoma (HCC) may develop on an NAFLD background. The authors unraveled, using microarrays and quantitative reverse-transcription polymerase chain reaction (qRT-PCR) in the livers of patients, a novel gene expression signature that independently correlated with NAFLD severity.1 Although these findings might help develop novel diagnostic tests and treatments that target NAFLD patients at greatest risk, there are some important aspects that need to be stressed. The choice of housekeeping (control) genes, whose expression is not susceptible to nutrients, is crucial to normalize results in gene expression studies involving liver tissue. Moylan et al. used ribosomal protein L35 (RPL35) as the only control gene for all analyses. Studies on the translatome (total proteins formed by mRNA translation) show tremendous fluctuations in polysome-associated RPL35 levels in the livers of obese versus chow-fed mice, and also between fasted and fed states.2 Gene expression of RPL35 is increased in HCC cell lines and tissues.3 Moylan et al. relied most likely on the fact that RPL35 levels did not vary significantly in the clinical samples analyzed. However, if the expression of a control gene is potentially heavily influenced by nutritional and proliferative status in the liver, that could pose a problem when defining a gene expression signature with clinical applications. There is now a strong consensus in the community on the validity of the “GeNorm” method, employing the geometric mean of multiple carefully selected housekeeping genes to achieve robust normalization of gene expression data.4, 5 It would be interesting to reassess the data obtained by Moylan et al. using three or more housekeeping genes for normalization. Without robust normalization strategies it will be hard to assess the prognostic power of gene expression signatures. Manlio Vinciguerra, Ph.D. University College London (UCL) Institute for Liver and Digestive Health UCL Medical School Royal Free Campus London, UK

Intercellular adhesion molecule 1 (ICAM-1) - A new substrate for the development of ventricular fibrillation?

Screening patients of an increased risk to develop ventricular fibrillation (VF) is of utmost medical interest [1], because VF might cause sudden cardiac death accounting for almost half of all cardiovascular deaths [2]. Primary VF most commonly occurs under circumstances of acute myocardial infarction (AMI) [3]. Clinical risk factors for the development of VF have been identified [4]. Contributing factors to VF such as genetic polymorphisms [5], inflammatory and coagulatory markers are increasingly focused [6–11]. The intercellular adhesion molecule 1 (ICAM-1) is located on the surface of white blood cells, macrophages, lymphocytes and endothelial cells and plays an important rolewithin inflammatory processes such as atherosclerosis [12,13]. Atherosclerosis develops as part of an inflammatory response either to endothelial cell injury or to the retention of subendothelial cholesterol-rich, atherogenic lipoproteins [14]. Specifically, interactions between neutrophils and the endothelium arise consisting in the rolling along the post-capillary venules and extravasation of leukocytes, which is stepwise mediated through selectins, integrins and ICAM molecules [15,16]. Soluble ICAM-1 (sICAM-1) in serum was shown to be up-regulated during AMI [17] and during ventricular reperfusion arrhythmias after coronary angioplasty [18]. For the present study, a possible subclinical inflammatory state, represented by the expression of ICAM-1mRNA of human blood cells or sICAM-1 in serum during follow-up after VF, was hypothesized in patients suffering from ischemia-related VF [11]. Within a retrospective observational study 90patientswithfirst AMI were included. 43 patients had VF (VF+) in the initial phase of AMI not related to coronary reperfusion. 47 patients presentedwithAMIwithout VF (VF−). Diagnosis of AMI was based on typical symptoms and signs according to ESC guidelines [19,20]. Coronary angiography was performed within 24 h after onset of symptoms. VF was either documented outside the hospital by paramedics or in the intensive care unit prior to percutaneous coronary intervention. Most of the patients with VF during AMI were resuscitated outside the hospital. Blood samples were taken remote from AMI with a median of 634 days. All patients were clinically asymptomatic at the time of blood sampling. Written informed consent was obtained from all patients. The study protocol was approved by the medical ethics commission II of theMedical FacultyMannheim, University of Heidelberg, Germany. RNA was prepared from whole blood containing all types of human blood cells (PAXgeneTM Blood tubes, PreAnalytiX, Germany). Quantification of ICAM-1 mRNA expression was done by real-time RT-PCR (RTqPCR) using the ΔΔCT method with GAPDH as housekeeping gene for normalization. Serum levels of sICAM-1 were measured by a commercially available enzyme-linked immunosorbent assay (Quantikine® Human sICAM-1/CD54 Immunoassay, R&D Systems). The calculations were performed with InStat (GraphPad, USA) and IBM SPSS (IBM SPSS, Germany). Patients' characteristics are summarized in Table 1. (VF+) patients revealed 48% lower ICAM-1 mRNA expression in human blood cells

Peripheral blood mRNA expression patterns to differentiate hepatocellular carcinoma from other hepatic diseases

Peripheral blood genes expressions profiling (GeXP) have been convinced to be more specific for the diagnosis of cancer and other diseases, and the GeXP system provides an ideal method to analyze multiple genes expression in one normalized and equable system. We aim to differentiate hepatocellular carcinoma from other hepatic diseases based on peripheral blood and the GeXP system. Fifteen selected hepatic diseases related genes with two house-keeping genes for normalization were detected by the GeXP system. The diagnosis model was based on K nearest neighbor classifier and cross validation, and software based on MATLAB software was built for differential diagnosis of hepatic diseases. Eight hepatic related genes were demonstrated to show an obvious statistic difference in expressions while the K nearest neighbors classifier showed that the accuracy for normal controls, hepatitis B, liver cirrhosis, hepatocellular carcinoma and the Other group was separately 80.57 %, 78.17 %, 84.48 %, 73.24 % and 85.85 %. The set of validation has been carried out to assess the accuracy of Model Two and the accuracy was even higher than the set of building for the model, except for the hepatitis B (HBV) group. A sensitive and specific GeXP system of eight genes has been developed for the accurate differential diagnosis of hepatic disease.

Peripheral blood mRNA expression patterns to differentiate hepatocellular carcinoma from other hepatic diseases

Peripheral blood genes expressions profiling (GeXP) have been convinced to be more specific for the diagnosis of cancer and other diseases, and the GeXP system provides an ideal method to analyze multiple genes expression in one normalized and equable system. We aim to differentiate hepatocellular carcinoma from other hepatic diseases based on peripheral blood and the GeXP system. Fifteen selected hepatic diseases related genes with two house-keeping genes for normalization were detected by the GeXP system. The diagnosis model was based on K nearest neighbor classifier and cross validation, and software based on MATLAB software was built for differential diagnosis of hepatic diseases. Eight hepatic related genes were demonstrated to show an obvious statistic difference in expressions while the K nearest neighbors classifier showed that the accuracy for normal controls, hepatitis B, liver cirrhosis, hepatocellular carcinoma and the Other group was separately 80.57 %, 78.17 %, 84.48 %, 73.24 % and 85.85 %. The set of validation has been carried out to assess the accuracy of Model Two and the accuracy was even higher than the set of building for the model, except for the hepatitis B (HBV) group. A sensitive and specific GeXP system of eight genes has been developed for the accurate differential diagnosis of hepatic disease.

In Acute Promyelocytic Leukemia (APL) Low BAALC Gene Expression Identifies a Group of Patients with Favorable Outcome in Overall Survival and Relapse Free Survival,

Abstract 3543 Introduction:Acute promyelocytic leukemia (APL) is characterized by the translocation t(15;17) which results in the PML-RARα fusion protein. ATRA based treatment regimens lead to long term survival in approximately 75% of cases. However, relapse occurs in about 15% of APL patients. Moreover, it is not yet known which underlying pathomechanisms drive relapse of the disease. In addition, to avoid over- or undertreatment of patients, respectively, risk stratification of patients according to biological or molecular criteria would most probably improve current treatment modalities. As opposed to other leukemias, in APL no molecular markers have been established for risk stratification as yet. The gene brain and acute leukemia, cytoplasmic (BAALC) has been shown to be of prognostic relevance in other leukemias such as T acute lymphoblastic leukemia (T-ALL) and acute myeloid leukemia (AML). Therefore, the aim of our study was to evaluate whether BAALC expression could be of prognostic value in APL.Materials and Methods: Bone marrow mononuclear cells were retrospectively evaluated in 86 APL patients after informed consent. 50 patients were female and 36 patients were male. Median age was 47 years (range: 19–82 years). All patients were diagnosed and treated in the German AML Cooperative Group (AMLCG) study. The treatment consisted of simultaneous ATRA and double induction (TAD/HAM) chemotherapy including high-dose ara-C, one cycle of consolidation chemotherapy (TAD) and 3 years monthly maintenance chemotherapy. BM cells were analyzed by multiplex reverse transcriptase quantitative real-time PCR (qRT-PCR) in triplicates on a LightCycler 480 (Roche, Mannheim, Germany). Glucose-6-phosphate-isomerase (GPI) served as a housekeeping gene for normalization. For quantification of relative expression values a modified delta-delta CT calculation model according to Pfaffl was used (Pfaffl MW, Nucleic Acids Res; 2001) after determination of PCR efficiencies. BAALC expression groups were defined as follows: BAALC expression lower than the 25th percentile (BAALClow) and higher than the 25th percentile (BAALC high). Time to complete remission, relapse free survival (RFS) and overall survival (OS) were calculated using the Kaplan-Meier method and a log-rank test was used to compare differences between the groups (p < 0.05). For statistical analysis the Statistical Analysis Software version 9.2 was used. Results:In univariate analysis low BAALC expression was significantly associated with a superior OS. Patients in the BAALClow group showed an OS at 10 years of 82% as compared to 60% in the BAALChigh group (p=0.025). Moreover, RFS in the BAALClow group was significantly higher at 10 years as compared to the BAALChigh group (86% vs. 59%; p=0.0087). Time to complete remission was not correlated to BAALC expression levels. Conclusion:Low BAALC expression identifies a group of APL patients with a highly superior outcome indicated by a better OS and RFS in these patients. However, these findings need to be validated in an independent prospective study and it should be investigated whether this marker is independent of the applied treatment regimen and other known relevant markers. However, since the translocation t(15;17) by itself is not able to induce leukemia BAALC might be an interesting target for further investigation. Disclosures:No relevant conflicts of interest to declare.

Silicon and Ralstonia solanacearum modulate expression stability of housekeeping genes in tomato

For analysis of gene expression profiles underlying tomato– Ralstonia solanacearum interaction and silicon-induced resistance, an internal control with high expression stability under the experimental conditions is needed as a prerequisite for accurate relative quantification of gene expression. Therefore, the expression stability of three housekeeping genes, phosphoglycerate kinase genes ( PGK), α-tubulin ( TUB) and actin ( ACT) in tomato under 24 experimental conditions including combinations of silicon amendments and inoculation with R. solanacearum at six time points was examined. The expression of ACT severely varied, in particular at the early phase after inoculation with the pathogen, suggesting a possible disabling the cytoskeleton that mediates resistance. Application of silicon resulted in more expression stability of the three housekeeping genes, showing alleviation of the biotic stress imposed by the pathogen. Although PGK and TUB showed reasonable expression stability, PGK was frequently ranked as the more stable among the treatments and over time. Thus, it is misleading to use a commonly used housekeeping gene for normalization without prior investigation of its expression stability. Here, we recommend to use either PGK alone, or PGK and TUB together as internal controls to increase the accuracy and to avoid error propagation in quantification of relative gene expression.

High WT1 Expression Is Associated with a Shorter Time to Complete Remission After ATRA Based Induction Therapy In Acute Promyelocytic Leukemia

AbstractAbstract 1694 Introduction:In acute promyelocytic leukemia (APL) the t(15;17) is detectable in the vast majority of cases and leads to the fusion transcript PML-RARα. The introduction of all-trans-retinoic acid (ATRA) and arsenic trioxide (ATO) into therapeutic management of APL resulted in long term remissions in about 80% of patients. However, relapse occurs in about 10–15% of cases and remains a major cause of treatment failure. The Wilms Tumor 1 gene (WT1) encodes for a transcription factor that is involved in normal and malignant hematopoiesis. Aberrant expression of WT1 has been shown to be associated with an adverse outcome in different types of hematological malignancies such as acute myeloid leukemia, acute lymphoblastic leukemia and myelodysplastic syndromes. To date only small series exist on aberrant WT1 expression in APL. Therefore, the aim of this study was to further elucidate the incidence of aberrant WT1 expression in APL and its prognostic significance for clinical endpoints. Methods:Bone marrow (BM) mononuclear cells were retrospectively evaluated in 89 APL patients (53 female, 36 male) and 21 healthy controls (15 female, 6 male) after informed consent. The median age of APL patients and healthy controls was 46 (range: 19–82 years) and 60 years (range: 21–84 years), respectively. All patients were diagnosed and treated in the German AML Cooperative Group (AMLCG) study. The treatment consisted of simultaneous ATRA and double induction (TAD/HAM) chemotherapy including high-dose ara-C, one cycle of consolidation chemotherapy (TAD) and 3 years monthly maintenance chemotherapy. BM cells were analyzed by multiplex reverse transcriptase quantitative real-time PCR (qRT-PCR) in triplicates on a LightCycler 480 (Roche, Mannheim, Germany). Beta–glucuronidase (GUS) served as a housekeeping gene for normalization. For quantification of relative expression values a modified delta-delta CT calculation model according to Pfaffl was used (Pfaffl MW, Nucleic Acids Res, 2001) after determination of PCR efficiencies. WT1 expression groups were defined as follows: WT1 expression higher than the 75th percentile (WT1high) and WT1 expression lower than the 25th percentile (WT1low). Time to complete remission, disease free survival (DFS) and overall survival (OS) were calculated using the Kaplan-Meier method and a log-rank test was used to compare differences between the groups (p < 0.05). Results:Normalized WT1 expression was significantly higher in APL samples (median: 0.1129, range: 0.00001–1.414) as compared to normal controls (median: 0.00135, range: 0.00001–0.01105) (p=0.0011). WT1high was not significantly associated with high white blood cell counts (WBC). In univariate analysis time to complete remission was significantly shorter in patients showing high WT1 expression as compared to patients with low WT1 expression (47 days vs. 58 days, p=0.0051). However, this did not translate into a longer DFS or OS. In contrast, there was a trend toward an impaired DFS and OS in the WT1high group. Interestingly, analyzing deaths prior to complete remission WT1high was significantly associated with early death as compared to the WT1low group (median 6 days vs. 17 days, p=0.0197). Conclusion:WT1 is highly expressed in APL as compared to healthy controls but is not associated with increased WBC. WT1high was significantly associated with a shorter time to CR. Since WT1 plays a critical role in differentiation and survival in myeolopoiesis this finding might be due to a higher succeptibility of WT1high APL patients to ATRA induced differentiation as compared to the WT1low group. Moreover, in vitro studies have shown that in NB4 cells WT1 mRNA levels decrease upon treatment with ATRA, this provides further evidence that WT1 might be an additional target for ATRA in APL (Gu W, Haematologica, 2005). Interestingly, WT1high is significantly correlated with early death prior to CR achievement. We hypothezise that this might also be an effect of an increased toxicity of the ATRA based induction therapy in patients having high WT1 expression. Disclosures:Lengfelder:Cephalon: Research Funding.

Evaluation of the transcription level of the protein disulfide isomerase in different stages from Ancylostoma caninum with a real-time PCR assay

The protein disulfide isomerase (PDI) is a ubiquitous protein, which contributes in building disulfide bridges. In the work presented here, the expression of the PDI in different stages of the canine hookworm Ancylostoma caninum was investigated. Third-stage larvae (L3), adults, as well as serum-stimulated and hypobiotic L3 were used. For quantification of the PDI gene transcription, a real-time PCR was used establishing a hybridization probe (TaqMantrade mark probes) for detection of PDI copy numbers in different populations. 18S ribosomal ribonucleic acid (rRNA) was used as a housekeeping gene for normalization. The results show differences in the transcription level of the investigated A. caninum populations: The serum-stimulated larvae representing the switch to parasitism showed the highest PDI expression. The hypobiotic larvae representing a resting stage showed the lowest expression level. Male adults showed an elevated expression compared to female adult worms. The L3 expression level was just below the serum-stimulated population. This work confirms the upregulated gene expression of PDI during host penetration and invasion.

Sodium Iodide Symporter Is Expressed at the Preneoplastic Stages of Liver Carcinogenesis and in Human Cholangiocarcinoma

The ability of thyroid cells to take up iodide, which enables (131)I radiotherapy for thyroid cancer, is due to the expression of the sodium iodide symporter at their plasma membrane. Expression of this symporter has been found in some nonthyroid cancers. However, it is mostly accumulated in the cytoplasm, and its functionality has not been demonstrated. We have investigated sodium iodide symporter expression and functionality in human liver cancer, and in a diethylnitrosamine induced Wistar rat model of primary liver cancer at different stages of carcinogenesis. Sodium iodide symporter mRNA and protein were studied in tissues from patients with hepatocellular- or cholangio-carcinomas using reverse-transcription polymerase chain reaction, immunoblot, and immunohistochemistry. We studied the dynamics of hepatic iodine uptake in the animal model using nuclear imaging. Sodium iodide symporter expression showed up in all 20 cholangiocarcinomas, but in only 2 of the 26 hepatocellular carcinomas, investigated. It was also found in normal bile duct cells and in the ductular reaction present in cirrhotic tissues. It was located at the plasma membrane in 10 of 20 cholangiocarcinoma. In rat liver cancer, a functional sodium iodide symporter expression was triggered as from the early preneoplastic steps, and was amplified during clonal tumor cell expansion, allowing complete tumor suppression after (131)I radiotherapy. A significant proportion of human cholangiocarcinomas expresses membrane sodium iodide symporter, which may permit radioiodine therapy. Our data also suggest that (131)I acts on a crucial target for liver cancer development.

Plasma membrane calcium ATPase (PMCA4): A housekeeper for RT-PCR relative quantification of polytopic membrane proteins

BackgroundAlthough relative quantification of real-time RT-PCR data can provide valuable information, one limitation remains the selection of an appropriate reference gene. No one gene has emerged as a universal reference gene and much debate surrounds some of the more commonly used reference genes, such as glyceraldehyde-3-phosphate dehydrogenase (GAPDH). At this time, no gene encoding for a plasma membrane protein serves as a reference gene, and relative quantification of plasma membrane proteins is performed with genes encoding soluble proteins, which differ greatly in quantity and in targeting and trafficking from plasma membrane proteins. In this work, our aim was to identify a housekeeping gene, ideally one that codes for a plasma membrane protein, whose expression remains the same regardless of drug treatment and across a wide range of tissues to be used for relative quantification of real-time RT-PCR data for ATP binding cassette (ABC) plasma membrane transporters.ResultsIn studies evaluating the expression levels of two commonly used reference genes coding for soluble proteins and two genes coding for membrane proteins, one plasma membrane protein, plasma membrane calcium-ATPase 4 (PMCA4), was comparable to the two reference genes already in use. In addition, PMCA4 expression shows little variation across eight drug-treated cell lines and was found to be superior to GAPDH and HPRT1, commonly used reference genes. Finally, we show PMCA4 used as a reference gene for normalizing ABC transporter expression in a drug-resistant lung carcinoma cell line.ConclusionWe have found that PMCA4 is a good housekeeping gene for normalization of gene expression for polytopic membrane proteins including transporters and receptors.