Simplified Fluorescent Multiplex PCR Method for Evaluation of the T-Cell Receptor V -Chain Repertoire

Size: px
Start display at page:

Download "Simplified Fluorescent Multiplex PCR Method for Evaluation of the T-Cell Receptor V -Chain Repertoire"

Transcription

1 CLINICAL AND DIAGNOSTIC LABORATORY IMMUNOLOGY, Apr. 2005, p Vol. 12, No X/05/$ doi: /cdli Copyright 2005, American Society for Microbiology. All Rights Reserved. Simplified Fluorescent Multiplex PCR Method for Evaluation of the T-Cell Receptor V -Chain Repertoire Sanjit Fernandes, Surendra Chavan, Vivek Chitnis, Nina Kohn, and Savita Pahwa* Immunology and Inflammation Center of Excellence, North Shore Long Island Jewish Research Institute, North Shore University Hospital NYU School of Medicine, Manhasset, NY Received 11 March 2004/Returned for modification 20 July 2004/Accepted 19 November 2004 Rationale: evaluation of the T-cell receptor (TCR) V -chain repertoire by PCR-based CDR3 length analysis allows fine resolution of the usage of the TCR V repertoire and is a sensitive tool to monitor changes in the T-cell compartment. A multiplex PCR method employing 24 labeled upstream V primers instead of the conventionally labeled downstream C primer is described. Method: RNA was isolated from purified CD4 and CD8 T-cell subsets from umbilical cord blood and clinical samples using TRI reagent followed by reverse transcription using a C primer and an Omniscript RT kit. The 24 V primers were multiplexed based on compatibility and product sizes into seven reactions. cdna was amplified using 24 V primers (labeled with tetrachloro-6-cardoxyfluorescein, 6-carboxyfluorescein, and hexachloro-6-carboxyfluorescein), an unlabeled C primer, and Taqgold polymerase. The fluorescent PCR products were resolved on an automated DNA sequencer and analyzed using the Genotyper 2.1 software. Results: V spectratypes of excellent resolution were obtained with RNA amounts of 250 ng using the labeled V primers. The resolution was superior to that obtained with the labeled C primer assay. Also the numbers of PCRs were reduced to 7 from the 12 required in the C labeling method, and the sample processing time was reduced by half. Conclusion: The method described for T-cell receptor V -chain repertoire analysis eliminates tedious dilutions and results in superior resolution with small amounts of RNA. The fast throughput makes this method suitable for automation and offers the feasibility to perform TCR V repertoire analyses in clinical trials. T cells recognize the antigen presented by antigen-presenting cells in the context of major histocompatibility complex class I (MHC I) and II molecules through the T-cell receptors (TCR) (4). The diversity in recognition of innumerable antigens is dependent on the diversity in TCR (3). TCR is a heterodimeric glycoprotein consisting of an alpha and beta chain (6). Each chain is the product of a complex gene recombination rearrangement process that takes place during the intrathymic differentiation (16). During recombination, TCR variable (V) diversity (D) and junctional (J) region segments are coupled to a constant (C) gene domain. The immense diversity created by these random recombination events and other processes, such as random nucleotide insertion, make the V-D-J region extremely variable in nature. The variable length of the CDR3 region is a function of the non-germ lineencoded event of nucleotide insertion by TdT and is the most hypervariable region of the -chain. It is this region that has been predicted to confer fine specificity of recognition to the TCR for the peptide-mhc complexes. Analysis of the CDR3 region of the TCR chain can thus provide insights into the heterogeneity of the T-cell compartment and of immune mechanisms operative in infectious and autoimmune diseases (18). The TCR V repertoire can be analyzed by different methods (2), including anchor PCR (12), heteroduplex PCR analysis (20, 21), and flow cytometry (10). Anchor PCR analysis amplifies the entire gene segment of the known and unknown families of * Corresponding author. Mailing address: University of Miami School of Medicine 712, Batchelor Children s Research Institute, University of Miami School of Medicine, 1580 N.W. 10th Avenue, Miami, FL Phone: (305) Fax: (305) the TCR repertoire but fails to resolve fine specificity of the CDR3 segment or the V gene usage. In the heteroduplex assay, the amplified cdna forms a duplex, and the output may compromise the fine specificity of each V family in comparison to that determined by the CDR3 length analysis. TCR repertoire analysis by flow cytometry utilizes monoclonal antibodies against the TCR chains and has the advantage of coupling the identification of TCR V families with phenotypic characterization of T cells. However, the method is limited by availability of monoclonal antibodies and its inability to determine diversity and restrictions in TCR gene usage, as is feasible by PCR analysis. The number of PCRs or probes needed to detect all V genes is a cumbersome feature of the assay, especially when multiple samples need to be analyzed. Several groups have applied the multiplex PCR method for the analysis of TCR genes (5, 7). Maslanka et al. (13) developed a system utilizing two specific T-cell receptor V -chain primers in each PCR for analysis of 23 T-cell receptor V -chain families coupled with spectratyping technology, and subsequently, a method (8, 14) that multiplexed two to three V primers with one primer specific for the TCR constant region was described. Another method (1) utilized a multiplex PCR system employing five PCRs combining 24 V primers. We have developed a multiplex PCR system that employs seven PCRs with four to six primers in each tube for the detection of the 24 V families. In this study we demonstrate the specificity and sensitivity of this multiplex system with a relatively low cell input and illustrate the resolution of the assay in patient samples. MATERIALS AND METHODS Reagents. Human anti-cd4/cd8 antibody-coated magnetic beads were purchased from Dynal Corp, Inc. (Great Neck, NY). Molecular biology grade Downloaded from on April 17, 2018 by guest 477

2 478 FERNANDES ET AL. CLIN. DIAGN. LAB. IMMUNOL. TABLE 1. Primers used for TCR V analysis using labeled V primers a Dye TCR V family Sequence Amplicon size (bp) Tube 6-FAM V 1 5 -CAA CAG TTC CCT GAC TTG CAC A V 4 5 -CAT ATG AGA GTG GAT TTG TCA TT G V 8 5 -TAC TTT AAC AAC AAC GTT CCG B V TCT CGA AAA GAG AAG AGG AAT F V GAG TCA GGA ATG CCA AAG GAA F V 6 5 -AGG CCT GAG GGA TCC GTC TC B V GAC AAA GGA GAA GTC TCA GAT C V ACA GTC TCC AGA ATA AGG ACG D V 3 5 -TCT AGA GAG AAG AAG GAG CGC E V GAT ATG AGA ATG AGG AAG CAG E TET V 2 5 -TCA ACC ATG CAA GCC TGA CCT A V 5S1 5 -TTC AGT GAG ACA CAG AGA AAC A V 7 5 -CTG AAT GCC CCA ACA GCT CTC B V 13S1 5 -GAC CAA GGA GAA GTC CCC AAT C V TCT GAG GTG CCC CAG AAT CTC D V CAC AGA TAG TAA ATG ACT TTC AG E V GAG TCT AAA CAG GAT GAG TCC B HEX V TCA TTT CGT TTT ATG AAA AGA TGC F V AAA GAT TTT AAC AAT GAA GCA GAC E V GTC TCT CGA CAG GCA CAG GCT D V CAG AGA AGT CTG AAA TAT TCG A G V 5S2 5 -CCT AAC TAT AGC TCT GAG CTG A V 9 5 -AAA TCT CCA GAC AAA GCT CAC B V 13S2 5 -GTT GGT GAG GGT ACA ACT GCC C None C -R 5 -CTT CTG ATG GCT CAA ACA C-3 a In this method the V primers were labeled with appropriate fluorescent tags as described in the text. For multiplexing primers see Materials and Methods. chloroform, isopropanol, dimethyl formamide, and TRI reagent were obtained from Sigma-Aldrich (St. Louis, MO). Glycoblue and Superasin were purchased from Ambion, Inc. (Austin, TX). An Omniscript reverse transcription (RT) kit was purchased from QIAGEN (Valencia, CA). AmplTaq Gold polymerase, deoxynucleoside triphosphates (dntps), internal size standard 6-carboxytetramethylrhodamine, AmpliTaq polymerase, dntp mix, and the fluorescent dyes 6-carboxyfluorescein (FAM), which is blue, tetrachloro-6-cardoxyfluorescein (TET), which is green, and hexachloro-6-carboxyfluorescein (HEX), which is yellow, were procured from PE Applied Biosystems (Foster City, CA). Samples. Umbilical cord blood samples were obtained under the guidelines of IRB-approved protocols from the North Shore Long Island Jewish Research Institute University Hospital in heparinized tubes within 4 h of delivery. Heparinized venous blood was also obtained from human immunodeficiency virus-positive (HIV ) patients engaged in clinical protocols under informed consent and from healthy volunteers. The cord blood mononuclear cells (CBMC) and peripheral blood mononuclear cell samples were isolated by standard Ficoll-Hypaque gradient centrifugation. CD4 and CD8 T cells were separated from cord blood mononuclear cells and peripheral blood mononuclear cells by positive selection using immunomagnetic beads coated with human anti-cd4 and anti-cd8 monoclonal antibodies (Dynal Corporation, Great Neck, NY), as previously described (2, 11, 19). RNA extraction and cdna synthesis. Total RNA was extracted from positively selected CD4 and CD8 T cells by a TRI reagent-chloroform extraction method (TRI reagent and molecular biology reagents, chloroform, isopropanol, and dimethyl formamide from Sigma, St. Louis, MO) according to the manufacturer s protocol. A total of 10 lof50 g/ml of glycoblue (Ambion, Austin, TX) was added to each ml of aqueous phase, and the RNA was subsequently precipitated with an equal volume of isopropanol. The precipitated RNA was dissolved in 10 l of DEPC-treated water containing 1U/ l of superasine (both from Ambion, Austin, TX). A total of 1 to g of RNA was reverse transcribed into cdna by using an Omniscript RT kit according to the manufacturer s protocol using an unlabeled C -Reverse primer (C -R) in a 40- l reaction mixture. Multiplex PCR primer combination. Table 1 lists the TCR V and the C -R primers used in this study. Locations of primers are shown in Fig. 1. Our first objective was to multiplex the V families into as few sets as possible without primer incompatibility and amplicon size differing in at least more than 10 nucleotides. Primer compatibility was analyzed using the Oligos 1998 to 2001 V.8.72 primer design freeware software available on the Internet site http: // (Institute of Biotechnology, University of Helsinki, Finland). Based on the amplicon size and primer compatibility, the V families were divided into three groups and labeled with fluorescent dyes FAM, TET, and HEX (Table 1). Multiplex PCR by labeled V primer. In this new method, seven set multiplex PCRs were carried out on a PTC-225 Peltier thermal cycler (MJ Research, San Francisco, CA). Primer sequences and how they were mixed are shown in Table 1. The primer set mixtures in tubes A to F were as follows: A (V 1, 2, 5.1, 5.2); B(V 6, 7, 8, 9, 16); C (V 12, 13.1, 13.2); D (V 11, 20, 15); E (V 3, 21, 17, 24); F (V 14, 18, 23); G (V 4, 22). Each multiplex reaction contained 1 l cdna, 1 l each of TCR V primer set specific for 3 to 5 different V families, and 2 M C -R, 1 mm dntps, 2 mm MgCl 2, and 1U of AmpliTaq Gold DNA polymerase in a final reaction mixture of 12.5 l. For the V primers 2, 7, 13.1, and 17, it was determined that the labeled primers had to be mixed with unlabeled primers at a ratio of 3:1 in order to prevent disproportionately high signal intensity. The PCR conditions were 94 C for 12 min for enzyme activation, followed by 35 cycles of 94 C for 20 s, 58 C for 20 s, and 72 C for 30 s, and finally one cycle of 72 C for 10 min. The titration of MgCl 2 and of each TCR V primer concentration was performed to optimize each multiplex reaction, to provide uniform amounts of amplified products. Multiplex PCR by labeled C primer. In this previously described conventional method (2, 11, 19), 12 reaction tubes were used with two to three primers in each tube. Multiplex PCRs were carried out as before on a PTC-225 Peltier thermal cycler. Analysis by spectratyping. The amplified PCR products were diluted 10 times with molecular biology grade water. The 2 l of the diluted product was mixed with 12 l of dimethyl formamide containing 0.5 l of Tamara 350 as internal size standard. The mixture was denatured at 95 C for 5 min and immediately cooled in an ice water bath for 5 min and resolved and size fractionated on an ABI 310 genetic analyzer (PE Biosystems, Foster City, CA). Raw data were further analyzed using a Genotyper 2.1 apparatus (PE Biosystems, Foster City, CA). RESULTS The spectratype of cord blood T cells determined by multiplex PCR exhibits a Gaussian pattern. The spectratype of Downloaded from on April 17, 2018 by guest

3 Downloaded from on April 17, 2018 by guest FIG. 1. TCR V repertoire in cord blood CD4 T cells, amplified by two different approaches. A, with labeled V forward primer and unlabeled C reverse primer; B, with unlabeled V forward primer and labeled C reverse primer. The resolution of TCR V families V 14, 15, 21, and V 24 is superior with labeled V primers. 479

4 480 FERNANDES ET AL. CLIN. DIAGN. LAB. IMMUNOL. Downloaded from FIG. 2. Frequency distribution histograms of TCR V families derived from normal umbilical cord blood T cells. The frequency probability distributions (y axis) of different CDR3 lengths (x axis) of individual TCR V families from 10 cord blood samples were averaged to generate a control composite profile for each V family, as described in the text. TCR V repertoire in cord blood CD4 T cells was performed using labeled V forward primers and unlabeled C reverse primer (Fig. 1A) and with unlabeled V forward primers and labeled C reverse primer, as illustrated in Fig. 1B. All the 24 known functional TCR V families could be amplified successfully from CD4 and CD8 T cells. Each V family is represented by a set of peaks, with each peak representing a set of T-cell clones bearing the same CDR3 length with different nucleotide sequences corresponding to different amino acid compositions. Within a TCR V family, the difference between each successive peak is three nucleotides, i.e., one amino acid. An unperturbed polyclonal repertoire for any V family is represented by a set of peaks distributed in a Gaussian pattern, with the highest-intensity CDR3 segment lying in the center. Deviation from this Gaussian pattern or reduction in the number of peaks is termed as perturbation and results either from overrepresentation, underrepresentation, or the absence of one or more CDR3 segments. Umbilical cord blood T cells have a relatively unperturbed repertoire and were used to create a standard profile against which the test sample is evaluated. Each cord blood V profile was measured as a frequency histogram and translated into a relative frequency probability distribution, with a total area equal to 1, as previously described (7). By averaging the frequency distributions obtained in 10 cord blood samples, a control composite profile was generated for each V family (Fig. 2). Deviation from the control profile for each V family in a test sample could theoretically range from 0 (complete overlap) to 100% (complete nonoverlap) perturbation. In order to assess the variation between the cord bloods themselves, percent perturbation was determined for each V family in individual cord blood T cells against the composite profile. Comparison of TCR V repertoire obtained using labeled C versus labeled V primers. Both methods provide good resolution of the different TCR transcripts. However, the multiplex PCR method performed by the labeled V forward primer method resulted in better resolution and higher signal intensities compared to that performed using labeled C reverse primers. The comparative analysis performed in 10 different CD4 and CD8 T cells isolated from 10 different cord blood revealed that the resolution of TCR V families V 14, 15, 21, and 24 improved significantly by using labeled V primers (Figs. 1A and 1B). The summary value of mean perturbations for all V families in cord blood was determined to on April 17, 2018 by guest

5 VOL. 12, 2005 TCR V REPERTOIRE 481 FIG. 3. RNA titration to determine the optimal input in the multiplex PCR using labeled V primers: amplification of the TCR V families at 250 ng RNA input is superior to that observed at an RNA input of 125 ng. Downloaded from be 8.83% with a standard deviation of 2.43 and an upper limit of 12.83% in the labeled V primer-7 tube method. For the 12 tube labeled C reverse primer method, the mean perturbation was 13.42% with a standard deviation of 1.48 and an upper limit of 15.86%. Importantly, the time and the reagents required for multiplex PCR performed by labeled V primers to amplify TCR V families is approximately half of that required for the multiplex PCR performed with the labeled C reverse primer. RNA titration to determine the sensitivity of multiplex PCR using labeled V primers. The RNA input required to obtain optimal resolution of the TCR V repertoire was determined using different concentrations of RNA ranging from 125 ng to 1,000 ng by double dilution in a 40- l RT reaction. The average yield of RNA was approximately 1.0 g per million cells. All TCR V families were amplified at 1,000 ng to 250 ng of starting RNA. As shown in Fig. 3, the TCR V resolution was compromised at 125 ng RNA, resulting in a perturbed spectratype pattern, compared to that obtained with RNA input of 250 ng. Analysis of TCR V repertoire in clinical samples. A control TCR V profile was developed using CD4 and CD8 T cells from normal umbilical cord blood samples to represent an unperturbed or naïve T-cell repertoire as described above. For each study subject, percent perturbations within each V family were computed after summing the absolute differences between each peak of the control profile and that of the study subject s profile. A single dominant peak constituting 50% of the total area of the other CDR3 segments in that particular V family was designated as dominant, which was previously shown to be clonal in nature by sequence analysis (14). The 7-tube method using the labeled V primers has recently been used for assessing perturbations in clinical samples from HIVinfected and uninfected adolescents (15). The percent perturbations in CD4 T cells were comparable to uninfected controls, but the incidence of clonal dominance was greater in patients CD4 T cells. In CD8 T cells, perturbations and clonal dominance in the infected patients was significantly greater than that of uninfected volunteers. Figure 4 is an example, showing the TCR V profiles of a subject with HIV infection. It can be noted that CD4 T cells showed perturbations in six TCR V families (mean perturbation, 15%), but almost all CD8 TCR V families were perturbed (mean perturbation, 29.5%) and five of them had a dominant profile. DISCUSSION The multiplex PCR using the labeled V forward primers described here was developed to facilitate the rapid qualitative analysis of the TCR V gene expression and to characterize alterations in the T-cell receptor repertoire. The method is very sensitive, is highly reproducible, and yields an improved resolution of the TCR V families in comparison to previously described methods. Additional advantages of this newly devel- on April 17, 2018 by guest

6 482 FERNANDES ET AL. CLIN. DIAGN. LAB. IMMUNOL. FIG. 4. Example of the TCR V repertoire in an HIV-infected patient using labeled V primers. The asterisks represent perturbations, and the arrows depict TCR V families exhibiting clonal dominance. oped method are the feasibility of using small patient samples and the speed with which it can be performed relative to the old method. A major requirement of TCR V repertoire studies is that of good quality cdna which is reverse transcribed from RNA that is isolated from highly purified populations of CD4 and CD8 T cells and their subsets. The amount of RNA in the reaction is thus an important factor for maintaining specificity, especially when analyzing T-cell receptor repertoire from various sources. Addition of an excessive amount of RNA can reduce the resolution of specific signals or generate spurious products. Excessive template is a potential cause for mispriming in allele-specific PCR (8). In developing this assay, the least amount of RNA that would yield optimal results was determined by titration of different concentrations of RNA (0.125 g to1.0 g) for the RT into cdna. RNA concentration of 0.25 g was found to be sufficient for qualitative analysis of the 24 V families. This feature makes the test feasible in situations where the clinical samples are limited, as in blood samples from infants and young children. Since each primer has different requirements for Mg and dntps and different affinities for the cognate target sequences, the optimal concentrations and number of reaction cycles required for proper amplification will differ. Optimizing these factors individually for each multiplex set or for each primer is not realistic in a system designed to handle a large number of samples. Titration of MgCl 2 (1.5 to 2 mm) revealed that 1.5 mm was best suited for the amplification in the multiplex PCR. Multiplex PCR can result in competition between different primers for templates, and thus, determination of intraprimer compatibility is necessary for achieving the best amplification efficiency of given primer pairs. Analysis of the TCR V repertoire using 100% labeled V primers revealed that some of the primers (V 2, 7, 13.1, and 17) were amplified at a significantly higher level compared to the others. The high intensities of these primers led them to bleed into the neighboring channels of the detector. This problem persisted even if the individual concentrations of the V primers were lowered, but it was overcome by adding corresponding unlabeled V primers, which competed with the labeled primers and resulted in lowering the signal intensity. For comparison between the assays using labeled V primers and the labeled C -R, we analyzed 10 cord blood samples by both methods. Cord blood provides an ideal source of an unperturbed TCR repertoire, because perturbation is a consequence of peripheral events (9). Moreover, it is fully diversified, as the genetic mechanisms for T-cell repertoire diversification are developed in the fetus by 24 weeks of gestation (17). The overall resolution of all the 24 V families was found to be superior with the use of labeled V primers in comparison to labeled C -R. This change in the method to using labeled V primers also decreased the assay steps and reduced the assay time almost by half. As shown in the example of the TCR V repertoire of one patient, this method of analysis provides excellent resolution of TCR V repertoire in clinical samples. It readily lends itself for studies requiring analysis of perturbations in sequential samples from the same patient, or between different patients, because the cord blood provides an unbiased standard that allows objective comparison of samples. Thus, the degree of perturbation in relation to the cord blood composite can be evaluated for each sample, allowing for objective analysis of clonal dominance and the change over time in each CDR3 length. This analysis can be performed in major subsets of T cells (e.g., in CD4 and CD8 T cells) as well as in functional subsets of CD4 and CD8 T cells, e.g., in naïve, memory, and effector cells. In conclusion, this method provides a tool for quantitative and qualitative assessment of the TCR V repertoire and can be used for few or many samples. The standardization procedure using the cord blood composite profile makes the results easier to interpret and to compare within and between subjects in longitudinal studies, making it feasible for analysis of TCR V repertoire in clinical trials. REFERENCES 1. Akatsuka, Y., E. G. Martin, A. Madonik, A. A. Barsoukov, and J. A. Hansen Rapid screening of T-cell receptor (TCR) variable gene usage by Downloaded from on April 17, 2018 by guest

7 VOL. 12, 2005 TCR V REPERTOIRE 483 multiplex PCR: application for assessment of clonal composition. Tissue Antigens 53: Chitnis, V., and S. Pahwa Evaluation of the T cell receptor repertoire, p In N. R. Rose, R. G. Hamilton, and B. Detrick (ed.), Manual of clinical laboratory immunology, 6th ed. ASM Press, Washington, D.C. 3. Davis, M., and M. Muller T cell receptor biochemistry, repertoire selection and general features of TCR and Ig structure. CIBA Found. Symp. 204: Ehrich, E. W., B.Devaux, E. P. Rock, J. L. Jorgensen, M. M. Davis, and Y. H. Chien T cell receptor interaction with peptide/major histocompatibility complex (MHC) and superantigen/mhc ligands is dominated by antigen. J. Exp. Med. 178: Fodinger, M., H. Buchmayer, I. Schwarzinger, I. Simonitsch, K. Winkler, U. Jager, R. Knobler, and C. Mannhalter Multiplex PCR for rapid detection of T-cell receptor-gamma chain gene rearrangements in patients with lymphoproliferative diseases. Br. J. Haematol. 94: Frank, M. M Structure/function analysis of the invariant subunits of the T cell antigen receptor. Semin. Immunol. 3: Gorochov, G., A. U. Neumann, A. Kereveur, C. Parizot, T. Li, C. Katlama, M. Karmochkine, G. Raguin, B. Austran, and P. Debre Perturbation of CD4 and CD8 T-cell repertoires during progression to AIDS and regulation of the CD4 repertoire during antiviral therapy. Nat. Med. 4: Gregersen, P. K., R. Hingorani, and J. Monteiro Oligoclonality in the CD8 T-cell population. Analysis using a multiplex PCR assay for CDR3 length. Ann. N. Y. Acad. Sci. 756: Halapi, E., M. Ehrani, A. Blucher, R. Andresson, P. Rossi, H. Wigzell, and J. Grunewald Diverse T-cell receptor CDR3 length patterns in human CD4 and CD8 T lymphocytes from newborns and adults. Scand. J. Immunol. 49: Kharbanda, M., T. W. McCloskey, R. Pahwa, and S. Pahwa Alterations in T-cell receptor V repertoire of CD8 T lymphocytes in HIVinfected children. Clin. Diagn. Lab. Immunol. 10: Kharbanda, M., S. Than, V. Chitnis, M. Sun, S. Chavan, S. Bakshi, and S. Pahwa Patterns of CD8 T cell clonal dominance and response to antiretroviral therapy in HIV infected children. AIDS 14: Loh, E. Y., J. F. Elliott, S. Cwirla, L. L. Lanier, and M. M. Davis Polymerase chain reaction with single-sided specificity: analysis of T cell receptor delta chain. Science 243: Maslanka, K., T. Pilatek, J. Gorski, M. Yassai, and J. Gorski Molecular analysis of T cell repertoires. Spectratypes generated by multiplex polymerase chain reaction and evaluated by radioactivity or fluorescence. Hum. Immunol. 44: Monteiro, J., R. Hingorani, I. H. Choi, J. Silver, R. Pergolizzi, and P. K. Gregersen Oligoclonality in the human CD8 T cell repertoire in normal subjects and monozygotic twins: implications for studies of infectious and autoimmune diseases. Mol. Med. 1: Pahwa, S., V. Chitnis, R. M. Mitchell, S. Fernandez, A. Chandrasekharan, C. M. Wilson, and S. D. Douglas CD4 and CD8 T cell receptor repertoire perturbations with normal levels of T cell receptor excision circles in HIV-infected, therapy-naive adolescents. Res. Hum. Retrovir. 19: Rosenberg, W. M., P. A. Moss, and J. I. Bell Variation in human T cell receptor V beta and J beta repertoire: analysis using anchor polymerase chain reaction. Eur. J. Immunol. 22: Schelonka, R. L., F. M. Raaphorst, D. Infante, E. Kraig, J. M. Teale, and A. J. Infante T cell receptor repertoire diversity and clonal expansion in human neonates. Pediatr. Res. 43: Soudeyns, H., P. Champagne, C. L. Holloway, G. U. Silvestri, N. Ringuette, J. Samson, N. Lapointe, and R. Sekaly Transient T cell receptor beta-chain variable region-specific expansions of CD4 and CD8 T cells during the early phase of pediatric human immunodeficiency virus infection: characterization of expanded cell populations by T cell receptor phenotyping. J. Infect. Dis. 181: Than, S., M. Kharbanda, V. Chitnis, S. Bakshi, P. K. Gregersen, and S. Pahwa Clonal dominance patterns of CD8 T cells in relation to disease progression in HIV-infected children. J. Immunol. 162: Uematsu, Y A novel and rapid cloning method for the T-cell receptor variable region sequences. Immunogenetics 34: Yoshioka, T., T. Matsutani, S. Iwagami, Y. Tsuruta, T. Kaneshige, T. Toyosaki, and R. Suzuki Quantitative analysis of the usage of human T cell receptor alpha and beta chain variable regions by reverse dot blot hybridization. J. Immunol. Methods 201: Downloaded from on April 17, 2018 by guest

Supplementary Table 3. 3 UTR primer sequences. Primer sequences used to amplify and clone the 3 UTR of each indicated gene are listed.

Supplementary Table 3. 3 UTR primer sequences. Primer sequences used to amplify and clone the 3 UTR of each indicated gene are listed. Supplemental Figure 1. DLKI-DIO3 mirna/mrna complementarity. Complementarity between the indicated DLK1-DIO3 cluster mirnas and the UTR of SOX2, SOX9, HIF1A, ZEB1, ZEB2, STAT3 and CDH1with mirsvr and PhastCons

More information

c Tuj1(-) apoptotic live 1 DIV 2 DIV 1 DIV 2 DIV Tuj1(+) Tuj1/GFP/DAPI Tuj1 DAPI GFP

c Tuj1(-) apoptotic live 1 DIV 2 DIV 1 DIV 2 DIV Tuj1(+) Tuj1/GFP/DAPI Tuj1 DAPI GFP Supplementary Figure 1 Establishment of the gain- and loss-of-function experiments and cell survival assays. a Relative expression of mature mir-484 30 20 10 0 **** **** NCP mir- 484P NCP mir- 484P b Relative

More information

Supplementary Appendix

Supplementary Appendix Supplementary Appendix This appendix has been provided by the authors to give readers additional information about their work. Supplement to: Sherman SI, Wirth LJ, Droz J-P, et al. Motesanib diphosphate

More information

Nature Structural & Molecular Biology: doi: /nsmb Supplementary Figure 1

Nature Structural & Molecular Biology: doi: /nsmb Supplementary Figure 1 Supplementary Figure 1 U1 inhibition causes a shift of RNA-seq reads from exons to introns. (a) Evidence for the high purity of 4-shU-labeled RNAs used for RNA-seq. HeLa cells transfected with control

More information

Supplemental Data. Shin et al. Plant Cell. (2012) /tpc YFP N

Supplemental Data. Shin et al. Plant Cell. (2012) /tpc YFP N MYC YFP N PIF5 YFP C N-TIC TIC Supplemental Data. Shin et al. Plant Cell. ()..5/tpc..95 Supplemental Figure. TIC interacts with MYC in the nucleus. Bimolecular fluorescence complementation assay using

More information

Table S1. Oligonucleotides used for the in-house RT-PCR assays targeting the M, H7 or N9. Assay (s) Target Name Sequence (5 3 ) Comments

Table S1. Oligonucleotides used for the in-house RT-PCR assays targeting the M, H7 or N9. Assay (s) Target Name Sequence (5 3 ) Comments SUPPLEMENTAL INFORMATION 2 3 Table S. Oligonucleotides used for the in-house RT-PCR assays targeting the M, H7 or N9 genes. Assay (s) Target Name Sequence (5 3 ) Comments CDC M InfA Forward (NS), CDC M

More information

a) Primary cultures derived from the pancreas of an 11-week-old Pdx1-Cre; K-MADM-p53

a) Primary cultures derived from the pancreas of an 11-week-old Pdx1-Cre; K-MADM-p53 1 2 3 4 5 6 7 8 9 10 Supplementary Figure 1. Induction of p53 LOH by MADM. a) Primary cultures derived from the pancreas of an 11-week-old Pdx1-Cre; K-MADM-p53 mouse revealed increased p53 KO/KO (green,

More information

Supplementary Document

Supplementary Document Supplementary Document 1. Supplementary Table legends 2. Supplementary Figure legends 3. Supplementary Tables 4. Supplementary Figures 5. Supplementary References 1. Supplementary Table legends Suppl.

More information

Abbreviations: P- paraffin-embedded section; C, cryosection; Bio-SA, biotin-streptavidin-conjugated fluorescein amplification.

Abbreviations: P- paraffin-embedded section; C, cryosection; Bio-SA, biotin-streptavidin-conjugated fluorescein amplification. Supplementary Table 1. Sequence of primers for real time PCR. Gene Forward primer Reverse primer S25 5 -GTG GTC CAC ACT ACT CTC TGA GTT TC-3 5 - GAC TTT CCG GCA TCC TTC TTC-3 Mafa cds 5 -CTT CAG CAA GGA

More information

Toluidin-Staining of mast cells Ear tissue was fixed with Carnoy (60% ethanol, 30% chloroform, 10% acetic acid) overnight at 4 C, afterwards

Toluidin-Staining of mast cells Ear tissue was fixed with Carnoy (60% ethanol, 30% chloroform, 10% acetic acid) overnight at 4 C, afterwards Toluidin-Staining of mast cells Ear tissue was fixed with Carnoy (60% ethanol, 30% chloroform, 10% acetic acid) overnight at 4 C, afterwards incubated in 100 % ethanol overnight at 4 C and embedded in

More information

Supplementary Figure 1. ROS induces rapid Sod1 nuclear localization in a dosagedependent manner. WT yeast cells (SZy1051) were treated with 4NQO at

Supplementary Figure 1. ROS induces rapid Sod1 nuclear localization in a dosagedependent manner. WT yeast cells (SZy1051) were treated with 4NQO at Supplementary Figure 1. ROS induces rapid Sod1 nuclear localization in a dosagedependent manner. WT yeast cells (SZy1051) were treated with 4NQO at different concentrations for 30 min and analyzed for

More information

Supplementary Figure 1 a

Supplementary Figure 1 a Supplementary Figure a Normalized expression/tbp (A.U.).6... Trip-br transcripts Trans Trans Trans b..5. Trip-br Ctrl LPS Normalized expression/tbp (A.U.) c Trip-br transcripts. adipocytes.... Trans Trans

More information

Supplementary Figures

Supplementary Figures Supplementary Figures Supplementary Figure 1. H3F3B expression in lung cancer. a. Comparison of H3F3B expression in relapsed and non-relapsed lung cancer patients. b. Prognosis of two groups of lung cancer

More information

Supplementary Materials

Supplementary Materials Supplementary Materials 1 Supplementary Table 1. List of primers used for quantitative PCR analysis. Gene name Gene symbol Accession IDs Sequence range Product Primer sequences size (bp) β-actin Actb gi

More information

Citation for published version (APA): Oosterveer, M. H. (2009). Control of metabolic flux by nutrient sensors Groningen: s.n.

Citation for published version (APA): Oosterveer, M. H. (2009). Control of metabolic flux by nutrient sensors Groningen: s.n. University of Groningen Control of metabolic flux by nutrient sensors Oosterveer, Maaike IMPORTANT NOTE: You are advised to consult the publisher's version (publisher's PDF) if you wish to cite from it.

More information

BIOLOGY 621 Identification of the Snorks

BIOLOGY 621 Identification of the Snorks Name: Date: Block: BIOLOGY 621 Identification of the Snorks INTRODUCTION: In this simulation activity, you will examine the DNA sequence of a fictitious organism - the Snork. Snorks were discovered on

More information

CD31 5'-AGA GAC GGT CTT GTC GCA GT-3' 5 ' -TAC TGG GCT TCG AGA GCA GT-3'

CD31 5'-AGA GAC GGT CTT GTC GCA GT-3' 5 ' -TAC TGG GCT TCG AGA GCA GT-3' Table S1. The primer sets used for real-time RT-PCR analysis. Gene Forward Reverse VEGF PDGFB TGF-β MCP-1 5'-GTT GCA GCA TGA ATC TGA GG-3' 5'-GGA GAC TCT TCG AGG AGC ACT T-3' 5'-GAA TCA GGC ATC GAG AGA

More information

Supplementary Figure 1 MicroRNA expression in human synovial fibroblasts from different locations. MicroRNA, which were identified by RNAseq as most

Supplementary Figure 1 MicroRNA expression in human synovial fibroblasts from different locations. MicroRNA, which were identified by RNAseq as most Supplementary Figure 1 MicroRNA expression in human synovial fibroblasts from different locations. MicroRNA, which were identified by RNAseq as most differentially expressed between human synovial fibroblasts

More information

Figure S1. Analysis of genomic and cdna sequences of the targeted regions in WT-KI and

Figure S1. Analysis of genomic and cdna sequences of the targeted regions in WT-KI and Figure S1. Analysis of genomic and sequences of the targeted regions in and indicated mutant KI cells, with WT and corresponding mutant sequences underlined. (A) cells; (B) K21E-KI cells; (C) D33A-KI cells;

More information

A smart acid nanosystem for ultrasensitive. live cell mrna imaging by the target-triggered intracellular self-assembly

A smart acid nanosystem for ultrasensitive. live cell mrna imaging by the target-triggered intracellular self-assembly Electronic Supplementary Material (ESI) for Chemical Science. This journal is The Royal Society of Chemistry 2017 A smart ZnO@polydopamine-nucleic acid nanosystem for ultrasensitive live cell mrna imaging

More information

SUPPLEMENTARY INFORMATION

SUPPLEMENTARY INFORMATION doi: 10.1038/nature05883 SUPPLEMENTARY INFORMATION Supplemental Figure 1 Prostaglandin agonists and antagonists alter runx1/cmyb expression. a-e, Embryos were exposed to (b) PGE2 and (c) PGI2 (20μM) and

More information

Supplementary Table 2. Conserved regulatory elements in the promoters of CD36.

Supplementary Table 2. Conserved regulatory elements in the promoters of CD36. Supplementary Table 1. RT-qPCR primers for CD3, PPARg and CEBP. Assay Forward Primer Reverse Primer 1A CAT TTG TGG CCT TGT GCT CTT TGA TGA GTC ACA GAA AGA ATC AAT TC 1B AGG AAA TGA ACT GAT GAG TCA CAG

More information

Plasmids Western blot analysis and immunostaining Flow Cytometry Cell surface biotinylation RNA isolation and cdna synthesis

Plasmids Western blot analysis and immunostaining Flow Cytometry Cell surface biotinylation RNA isolation and cdna synthesis Plasmids psuper-retro-s100a10 shrna1 was constructed by cloning the dsdna oligo 5 -GAT CCC CGT GGG CTT CCA GAG CTT CTT TCA AGA GAA GAA GCT CTG GAA GCC CAC TTT TTA-3 and 5 -AGC TTA AAA AGT GGG CTT CCA GAG

More information

Beta Thalassemia Sami Khuri Department of Computer Science San José State University Spring 2015

Beta Thalassemia Sami Khuri Department of Computer Science San José State University Spring 2015 Bioinformatics in Medical Product Development SMPD 287 Three Beta Thalassemia Sami Khuri Department of Computer Science San José State University Hemoglobin Outline Anatomy of a gene Hemoglobinopathies

More information

Nature Immunology: doi: /ni.3836

Nature Immunology: doi: /ni.3836 Supplementary Figure 1 Recombinant LIGHT-VTP induces pericyte contractility and endothelial cell activation. (a) Western blot showing purification steps for full length murine LIGHT-VTP (CGKRK) protein:

More information

Supplementary Figure 1

Supplementary Figure 1 Supplementary Figure 1 Supplementary Figure 1: Cryopreservation alters CD62L expression by CD4 T cells. Freshly isolated (left) or cryopreserved PBMCs (right) were stained with the mix of antibodies described

More information

Beta Thalassemia Case Study Introduction to Bioinformatics

Beta Thalassemia Case Study Introduction to Bioinformatics Beta Thalassemia Case Study Sami Khuri Department of Computer Science San José State University San José, California, USA sami.khuri@sjsu.edu www.cs.sjsu.edu/faculty/khuri Outline v Hemoglobin v Alpha

More information

Culture Density (OD600) 0.1. Culture Density (OD600) Culture Density (OD600) Culture Density (OD600) Culture Density (OD600)

Culture Density (OD600) 0.1. Culture Density (OD600) Culture Density (OD600) Culture Density (OD600) Culture Density (OD600) A. B. C. D. E. PA JSRI JSRI 2 PA DSAM DSAM 2 DSAM 3 PA LNAP LNAP 2 LNAP 3 PAO Fcor Fcor 2 Fcor 3 PAO Wtho Wtho 2 Wtho 3 Wtho 4 DTSB Low Iron 2 4 6 8 2 4 6 8 2 22 DTSB Low Iron 2 4 6 8 2 4 6 8 2 22 DTSB

More information

Supplementary Figure 1

Supplementary Figure 1 Metastatic melanoma Primary melanoma Healthy human skin Supplementary Figure 1 CD22 IgG4 Supplementary Figure 1: Immunohisochemical analysis of CD22+ (left) and IgG4 (right), cells (shown in red and indicated

More information

www.lessonplansinc.com Topic: Protein Synthesis - Sentence Activity Summary: Students will simulate transcription and translation by building a sentence/polypeptide from words/amino acids. Goals & Objectives:

More information

Supplementary Figure 1

Supplementary Figure 1 Supplementary Figure 1 Supplementary Figure 1. Lats1/2 deleted ihbs and ihps showed decreased transcripts of hepatocyte related genes (a and b) Western blots (a) and recombination PCR (b) of control and

More information

Astaxanthin prevents and reverses diet-induced insulin resistance and. steatohepatitis in mice: A comparison with vitamin E

Astaxanthin prevents and reverses diet-induced insulin resistance and. steatohepatitis in mice: A comparison with vitamin E Supplementary Information Astaxanthin prevents and reverses diet-induced insulin resistance and steatohepatitis in mice: A comparison with vitamin E Yinhua Ni, 1,2 Mayumi Nagashimada, 1 Fen Zhuge, 1 Lili

More information

Expression of Selected Inflammatory Cytokine Genes in Bladder Biopsies

Expression of Selected Inflammatory Cytokine Genes in Bladder Biopsies Borneo Journal of Resource Science and Technology (2013) 3(2): 15-20 Expression of Selected Inflammatory Cytokine Genes in Bladder Biopsies EDMUND UI-HANG SIM *1, NUR DIANA ANUAR 2, TENG-AIK ONG 3, GUAN-

More information

Supplemental Information. Th17 Lymphocytes Induce Neuronal. Cell Death in a Human ipsc-based. Model of Parkinson's Disease

Supplemental Information. Th17 Lymphocytes Induce Neuronal. Cell Death in a Human ipsc-based. Model of Parkinson's Disease Cell Stem Cell, Volume 23 Supplemental Information Th17 Lymphocytes Induce Neuronal Cell Death in a Human ipsc-based Model of Parkinson's Disease Annika Sommer, Franz Maxreiter, Florian Krach, Tanja Fadler,

More information

Supplementary Materials and Methods

Supplementary Materials and Methods DD2 suppresses tumorigenicity of ovarian cancer cells by limiting cancer stem cell population Chunhua Han et al. Supplementary Materials and Methods Analysis of publicly available datasets: To analyze

More information

Supplementary Figure 1a

Supplementary Figure 1a Supplementary Figure 1a Hours: E-cadherin TGF-β On TGF-β Off 0 12 24 36 48 24 48 72 Vimentin βactin Fig. S1a. Treatment of AML12 cells with TGF-β induces EMT. Treatment of AML12 cells with TGF-β results

More information

Nucleotide Sequence of the Australian Bluetongue Virus Serotype 1 RNA Segment 10

Nucleotide Sequence of the Australian Bluetongue Virus Serotype 1 RNA Segment 10 J. gen. Virol. (1988), 69, 945-949. Printed in Great Britain 945 Key words: BTV/genome segment lo/nucleotide sequence Nucleotide Sequence of the Australian Bluetongue Virus Serotype 1 RNA Segment 10 By

More information

BHP 2-7 and Nthy-ori 3-1 cells were grown in RPMI1640 medium (Hyclone) supplemented with 10% fetal bovine serum (Gibco), 2mM L-glutamine, and 100 U/mL

BHP 2-7 and Nthy-ori 3-1 cells were grown in RPMI1640 medium (Hyclone) supplemented with 10% fetal bovine serum (Gibco), 2mM L-glutamine, and 100 U/mL 1 2 3 4 Materials and Methods Cell culture BHP 2-7 and Nthy-ori 3-1 cells were grown in RPMI1640 medium (Hyclone) 5 supplemented with 10% fetal bovine serum (Gibco), 2mM L-glutamine, and 100 U/mL 6 penicillin-streptomycin.

More information

Cross-talk between mineralocorticoid and angiotensin II signaling for cardiac

Cross-talk between mineralocorticoid and angiotensin II signaling for cardiac ONLINE SUPPLEMENT TO Crosstalk between mineralocorticoid and angiotensin II signaling for cardiac remodeling An Di ZHANG,,3, Aurelie NGUYEN DINH CAT*,,3, Christelle SOUKASEUM *,,3, Brigitte ESCOUBET, 4,

More information

HCV Persistence and Immune Evasion in the Absence of Memory T Cell Help.

HCV Persistence and Immune Evasion in the Absence of Memory T Cell Help. SOM Text HCV Persistence and Immune Evasion in the Absence of Memory T Cell Help. Arash Grakoui 1, Naglaa H. Shoukry 2, David J. Woollard 2, Jin-Hwan Han 1, Holly L. Hanson 1, John Ghrayeb 3, Krishna K.

More information

CIRCRESAHA/2004/098145/R1 - ONLINE 1. Validation by Semi-quantitative Real-Time Reverse Transcription PCR

CIRCRESAHA/2004/098145/R1 - ONLINE 1. Validation by Semi-quantitative Real-Time Reverse Transcription PCR CIRCRESAHA/2004/098145/R1 - ONLINE 1 Expanded Materials and Methods Validation by Semi-quantitative Real-Time Reverse Transcription PCR Expression patterns of 13 genes (Online Table 2), selected with respect

More information

Supplementary Figure 1

Supplementary Figure 1 Supplementary Figure 1 3 3 3 1 1 Bregma -1.6mm 3 : Bregma Ref) Http://www.mbl.org/atlas165/atlas165_start.html Bregma -.18mm Supplementary Figure 1 Schematic representation of the utilized brain slice

More information

Resistance to Tetracycline Antibiotics by Wangrong Yang, Ian F. Moore, Kalinka P. Koteva, Donald W. Hughes, David C. Bareich and Gerard D. Wright.

Resistance to Tetracycline Antibiotics by Wangrong Yang, Ian F. Moore, Kalinka P. Koteva, Donald W. Hughes, David C. Bareich and Gerard D. Wright. Supplementary Data for TetX is a Flavin-Dependent Monooxygenase Conferring Resistance to Tetracycline Antibiotics by Wangrong Yang, Ian F. Moore, Kalinka P. Koteva, Donald W. Hughes, David C. Bareich and

More information

*To whom correspondence should be addressed. This PDF file includes:

*To whom correspondence should be addressed.   This PDF file includes: www.sciencemag.org/cgi/content/full/science.1212182/dc1 Supporting Online Material for Partial Retraction to Detection of an Infectious Retrovirus, XMRV, in Blood Cells of Patients with Chronic Fatigue

More information

Supplemental Information. Cancer-Associated Fibroblasts Neutralize. the Anti-tumor Effect of CSF1 Receptor Blockade

Supplemental Information. Cancer-Associated Fibroblasts Neutralize. the Anti-tumor Effect of CSF1 Receptor Blockade Cancer Cell, Volume 32 Supplemental Information Cancer-Associated Fibroblasts Neutralize the Anti-tumor Effect of CSF1 Receptor Blockade by Inducing PMN-MDSC Infiltration of Tumors Vinit Kumar, Laxminarasimha

More information

Cancer Genetics 204 (2011) 45e52

Cancer Genetics 204 (2011) 45e52 Cancer Genetics 204 (2011) 45e52 Exon scanning by reverse transcriptaseepolymerase chain reaction for detection of known and novel EML4eALK fusion variants in nonesmall cell lung cancer Heather R. Sanders

More information

Lezione 10. Sommario. Bioinformatica. Lezione 10: Sintesi proteica Synthesis of proteins Central dogma: DNA makes RNA makes proteins Genetic code

Lezione 10. Sommario. Bioinformatica. Lezione 10: Sintesi proteica Synthesis of proteins Central dogma: DNA makes RNA makes proteins Genetic code Lezione 10 Bioinformatica Mauro Ceccanti e Alberto Paoluzzi Lezione 10: Sintesi proteica Synthesis of proteins Dip. Informatica e Automazione Università Roma Tre Dip. Medicina Clinica Università La Sapienza

More information

Description of Supplementary Files. File Name: Supplementary Information Description: Supplementary Figures and Supplementary Tables

Description of Supplementary Files. File Name: Supplementary Information Description: Supplementary Figures and Supplementary Tables Description of Supplementary Files File Name: Supplementary Information Description: Supplementary Figures and Supplementary Tables Supplementary Figure 1: (A), HCT116 IDH1-WT and IDH1-R132H cells were

More information

Phylogenetic analysis of human and chicken importins. Only five of six importins were studied because

Phylogenetic analysis of human and chicken importins. Only five of six importins were studied because Supplementary Figure S1 Phylogenetic analysis of human and chicken importins. Only five of six importins were studied because importin-α6 was shown to be testis-specific. Human and chicken importin protein

More information

TetR repressor-based bioreporters for the detection of doxycycline using Escherichia

TetR repressor-based bioreporters for the detection of doxycycline using Escherichia Supplementary materials TetR repressor-based bioreporters for the detection of doxycycline using Escherichia coli and Acinetobacter oleivorans Hyerim Hong and Woojun Park * Department of Environmental

More information

Supplementary Information. Bamboo shoot fiber prevents obesity in mice by. modulating the gut microbiota

Supplementary Information. Bamboo shoot fiber prevents obesity in mice by. modulating the gut microbiota Supplementary Information Bamboo shoot fiber prevents obesity in mice by modulating the gut microbiota Xiufen Li 1,2, Juan Guo 1, Kailong Ji 1,2, and Ping Zhang 1,* 1 Key Laboratory of Tropical Plant Resources

More information

Relationship of the APOA5/A4/C3/A1 gene cluster and APOB gene polymorphisms with dyslipidemia

Relationship of the APOA5/A4/C3/A1 gene cluster and APOB gene polymorphisms with dyslipidemia elationship of the APOA5/A4/C3/A1 gene cluster and APOB gene polymorphisms with dyslipidemia H.J. Ou 1, G. Huang 2, W. Liu 3, X.L. Ma 2, Y. Wei 4, T. Zhou 5 and Z.M. Pan 3 1 Department of Neurology, The

More information

The Clinical Performance of Primary HPV Screening, Primary HPV Screening Plus Cytology Cotesting, and Cytology Alone at a Tertiary Care Hospital

The Clinical Performance of Primary HPV Screening, Primary HPV Screening Plus Cytology Cotesting, and Cytology Alone at a Tertiary Care Hospital The Clinical Performance of Primary HPV Screening, Primary HPV Screening Plus Cytology Cotesting, and Cytology Alone at a Tertiary Care Hospital Jung-Woo Choi MD, PhD; Younghye Kim MD, PhD; Ju-Han Lee

More information

A basic helix loop helix transcription factor controls cell growth

A basic helix loop helix transcription factor controls cell growth A basic helix loop helix transcription factor controls cell growth and size in root hairs Keke Yi 1,2, Benoît Menand 1,3, Elizabeth Bell 1, Liam Dolan 1,4 Supplementary note Low soil phosphate availability

More information

SUPPORTING INFORMATION

SUPPORTING INFORMATION SUPPORTING INFORMATION Biology is different in small volumes: endogenous signals shape phenotype of primary hepatocytes cultured in microfluidic channels Amranul Haque, Pantea Gheibi, Yandong Gao, Elena

More information

Supporting Information

Supporting Information Supporting Information Malapeira et al. 10.1073/pnas.1217022110 SI Materials and Methods Plant Material and Growth Conditions. A. thaliana seedlings were stratified at 4 C in the dark for 3 d on Murashige

More information

Baseline clinical characteristics for the 81 CMML patients Routine diagnostic testing and statistical analyses... 3

Baseline clinical characteristics for the 81 CMML patients Routine diagnostic testing and statistical analyses... 3 Next-Generation Sequencing Technology Reveals a Characteristic Pattern of Molecular Mutations in 72.8% of Chronic Myelomonocytic Leukemia (CMML) by Detecting Frequent Alterations in TET2, CBL, RAS, and

More information

SUPPLEMENTARY DATA. Supplementary Table 1. Primer sequences for qrt-pcr

SUPPLEMENTARY DATA. Supplementary Table 1. Primer sequences for qrt-pcr Supplementary Table 1. Primer sequences for qrt-pcr Gene PRDM16 UCP1 PGC1α Dio2 Elovl3 Cidea Cox8b PPARγ AP2 mttfam CyCs Nampt NRF1 16s-rRNA Hexokinase 2, intron 9 β-actin Primer Sequences 5'-CCA CCA GCG

More information

Characterizing intra-host influenza virus populations to predict emergence

Characterizing intra-host influenza virus populations to predict emergence Characterizing intra-host influenza virus populations to predict emergence June 12, 2012 Forum on Microbial Threats Washington, DC Elodie Ghedin Center for Vaccine Research Dept. Computational & Systems

More information

Mutation Screening and Association Studies of the Human UCP 3 Gene in Normoglycemic and NIDDM Morbidly Obese Patients

Mutation Screening and Association Studies of the Human UCP 3 Gene in Normoglycemic and NIDDM Morbidly Obese Patients Mutation Screening and Association Studies of the Human UCP 3 Gene in Normoglycemic and NIDDM Morbidly Obese Patients Shuichi OTABE, Karine CLEMENT, Séverine DUBOIS, Frederic LEPRETRE, Veronique PELLOUX,

More information

Single-Molecule Analysis of Gene Expression Using Two-Color RNA- Labeling in Live Yeast

Single-Molecule Analysis of Gene Expression Using Two-Color RNA- Labeling in Live Yeast Supplemental Figures, Tables and Results Single-Molecule Analysis of Gene Expression Using Two-Color RNA- Labeling in Live Yeast Sami Hocine 1, Pascal Raymond 2, Daniel Zenklusen 2, Jeffrey A. Chao 1 &

More information

SUPPLEMENTAL METHODS Cell culture RNA extraction and analysis Immunohistochemical analysis and laser capture microdissection (LCM)

SUPPLEMENTAL METHODS Cell culture RNA extraction and analysis Immunohistochemical analysis and laser capture microdissection (LCM) SUPPLEMENTAL METHODS Cell culture Human peripheral blood mononuclear cells were isolated from healthy donors by Ficoll density gradient centrifugation. Monocyte differentiation to resting macrophages ()

More information

Oligo Sequence* bp %GC Tm Hair Hm Ht Position Size Ref. HIVrt-F 5 -CTA-gAA-CTT-TRA-ATg-CAT-ggg-TAA-AAg-TA

Oligo Sequence* bp %GC Tm Hair Hm Ht Position Size Ref. HIVrt-F 5 -CTA-gAA-CTT-TRA-ATg-CAT-ggg-TAA-AAg-TA Human immunodeficiency virus (HIV) detection & quantitation by qrt-pcr (Taqman). Created on: Oct 26, 2010; Last modified by: Jul 17, 2017; Version: 3.0 This protocol describes the qrt-pcr taqman based

More information

Journal of Cell Science Supplementary information. Arl8b +/- Arl8b -/- Inset B. electron density. genotype

Journal of Cell Science Supplementary information. Arl8b +/- Arl8b -/- Inset B. electron density. genotype J. Cell Sci. : doi:.4/jcs.59: Supplementary information E9. A Arl8b /- Arl8b -/- Arl8b Arl8b non-specific band Gapdh Tbp E7.5 HE Inset B D Control al am hf C E Arl8b -/- al am hf E8.5 F low middle high

More information

Isolate Sexual Idiomorph Species

Isolate Sexual Idiomorph Species SUPLEMENTARY TABLE 1. Isolate identification, sexual idiomorph and species of each isolate used for MAT locus distribution in Paracoccidioides species. Isolate Sexual Idiomorph Species Pb01 MAT1-1 P. lutzii

More information

Detection of 549 new HLA alleles in potential stem cell donors from the United States, Poland and Germany

Detection of 549 new HLA alleles in potential stem cell donors from the United States, Poland and Germany HLA ISSN 2059-2302 BRIEF COMMUNICATION Detection of 549 new HLA alleles in potential stem cell donors from the United States, Poland and Germany C. J. Hernández-Frederick 1, N. Cereb 2,A.S.Giani 1, J.

More information

Supplementary information

Supplementary information Supplementary information Unique polypharmacology nuclear receptor modulator blocks inflammatory signaling pathways Mi Ra Chang 1, Anthony Ciesla 1, Timothy S. Strutzenberg 1, Scott J. Novick 1, Yuanjun

More information

Supplemental Figures: Supplemental Figure 1

Supplemental Figures: Supplemental Figure 1 Supplemental Figures: Supplemental Figure 1 Suppl. Figure 1. BM-DC infection with H. pylori does not induce cytotoxicity and treatment of BM-DCs with H. pylori sonicate, but not heat-inactivated bacteria,

More information

University of Groningen. Vasoregression in incipient diabetic retinopathy Pfister, Frederick

University of Groningen. Vasoregression in incipient diabetic retinopathy Pfister, Frederick University of Groningen Vasoregression in incipient diabetic retinopathy Pfister, Frederick IMPORTANT NOTE: You are advised to consult the publisher's version (publisher's PDF) if you wish to cite from

More information

Supporting Information. Mutational analysis of a phenazine biosynthetic gene cluster in

Supporting Information. Mutational analysis of a phenazine biosynthetic gene cluster in Supporting Information for Mutational analysis of a phenazine biosynthetic gene cluster in Streptomyces anulatus 9663 Orwah Saleh 1, Katrin Flinspach 1, Lucia Westrich 1, Andreas Kulik 2, Bertolt Gust

More information

A Rapid and Sensitive Chip-based Assay for Detection of rpob Gene Mutations Conferring Rifampicin Resistance in Mycobacterium tuberculosis (TB).

A Rapid and Sensitive Chip-based Assay for Detection of rpob Gene Mutations Conferring Rifampicin Resistance in Mycobacterium tuberculosis (TB). A Rapid and Sensitive Chip-based Assay for Detection of rpob Gene Mutations Conferring Rifampicin Resistance in Mycobacterium tuberculosis (TB). Wanyuan Ao, Steve Aldous, Evelyn Woodruff, Brian Hicke,

More information

What do you think of when you here the word genome?

What do you think of when you here the word genome? What do you think of when you here the word genome? What do you think of when you here the word genome? Personal Genomics Outline Review of pre-lab work Genomics and Medicine Case Overview & Assignment

More information

Frequency of mosaicism points towards mutation prone early cleavage cell divisions.

Frequency of mosaicism points towards mutation prone early cleavage cell divisions. Frequency of mosaicism points towards mutation prone early cleavage cell divisions. Chad Harland, Wouter Coppieters, Latifa Karim, Carole Charlier, Michel Georges Germ-line de novo mutations Definition:

More information

Enhanced detection and serotyping of Streptococcus pneumoniae using multiplex polymerase chain reaction

Enhanced detection and serotyping of Streptococcus pneumoniae using multiplex polymerase chain reaction Original article http://dx.doi.org/10.3345/kjp.2012.55.11.424 Korean J Pediatr 2012;55(11):424-429 eissn 1738-1061 pissn 2092-7258 Enhanced detection and serotyping of Streptococcus pneumoniae using multiplex

More information

Supplementary Information

Supplementary Information Supplementary Information Remodeling of heterochromatin structure slows neuropathological progression and prolongs survival in an animal model of Huntington s disease Junghee Lee, Yu Jin Hwang, Yunha Kim,

More information

ice-cold 70% ethanol with gentle vortexing, incubated at -20 C for 4 hours, and washed with PBS.

ice-cold 70% ethanol with gentle vortexing, incubated at -20 C for 4 hours, and washed with PBS. Cell cycle analysis For cell cycle analysis, single cell suspensions of E12.5 fetal liver cells were suspended in 4 ml ice-cold 7% ethanol with gentle vortexing, incubated at -2 C for 4 hours, and washed

More information

Recommended laboratory tests to identify influenza A/H5 virus in specimens from patients with an influenza-like illness

Recommended laboratory tests to identify influenza A/H5 virus in specimens from patients with an influenza-like illness World Health Organization Recommended laboratory tests to identify influenza A/H5 virus in specimens from patients with an influenza-like illness General information Highly pathogenic avian influenza (HPAI)

More information

without LOI phenotype by breeding female wild-type C57BL/6J and male H19 +/.

without LOI phenotype by breeding female wild-type C57BL/6J and male H19 +/. Sakatani et al. 1 Supporting Online Material Materials and methods Mice and genotyping: H19 mutant mice with C57BL/6J background carrying a deletion in the structural H19 gene (3 kb) and 10 kb of 5 flanking

More information

Viral hepatitis, which affects half a billion people

Viral hepatitis, which affects half a billion people GASTROENTEROLOGY 2006;130:435 452 BASIC LIVER, PANCREAS, AND BILIARY TRACT Natural Killer Cells Ameliorate Liver Fibrosis by Killing Activated Stellate Cells in NKG2D-Dependent and Tumor Necrosis Factor

More information

Mechanistic and functional insights into fatty acid activation in Mycobacterium tuberculosis SUPPLEMENTARY INFORMATION

Mechanistic and functional insights into fatty acid activation in Mycobacterium tuberculosis SUPPLEMENTARY INFORMATION Mechanistic and functional insights into fatty acid activation in Mycobacterium tuberculosis Pooja Arora 1, Aneesh Goyal 2, Vivek T atarajan 1, Eerappa Rajakumara 2, Priyanka Verma 1, Radhika Gupta 3,

More information

Nucleotide diversity of the TNF gene region in an African village

Nucleotide diversity of the TNF gene region in an African village (2001) 2, 343 348 2001 Nature Publishing Group All rights reserved 1466-4879/01 $15.00 www.nature.com/gene Nucleotide diversity of the TNF gene region in an African village A Richardson 1, F Sisay-Joof

More information

Advanced Subsidiary Unit 1: Lifestyle, Transport, Genes and Health

Advanced Subsidiary Unit 1: Lifestyle, Transport, Genes and Health Write your name here Surname Other names Edexcel GCE Centre Number Candidate Number Biology Advanced Subsidiary Unit 1: Lifestyle, Transport, Genes and Health Thursday 8 January 2009 Morning Time: 1 hour

More information

Development of RT-qPCR-based molecular diagnostic assays for therapeutic target selection of breast cancer patients

Development of RT-qPCR-based molecular diagnostic assays for therapeutic target selection of breast cancer patients Development of RT-qPCR-based molecular diagnostic assays for therapeutic target selection of breast cancer patients Sangjung Park The Graduate School Yonsei University Department of Biomedical Laboratory

More information

SUPPLEMENTARY INFORMATION

SUPPLEMENTARY INFORMATION SUPPLEMENTARY INFORMATION Autonomous Multistep Organic Synthesis in a Single Isothermal Solution Mediated by a DNA Walker Yu He and David R. Liu* Supplementary Methods General Methods. DNA oligonucleotides

More information

SUPPLEMENTARY INFORMATION

SUPPLEMENTARY INFORMATION BASELINE ISCHAEMIA a b Phd2 +/- c d Collateral growth and maintenance SMC recruitment SMC proliferation Phd2 +/- NF- B off NF- B on NF- B on NF- B on Endothelial cell Smooth muscle cell Pro-arteriogenic

More information

Formylpeptide receptor2 contributes to colon epithelial homeostasis, inflammation, and tumorigenesis

Formylpeptide receptor2 contributes to colon epithelial homeostasis, inflammation, and tumorigenesis Supplementary Data Formylpeptide receptor2 contributes to colon epithelial homeostasis, inflammation, and tumorigenesis Keqiang Chen, Mingyong Liu, Ying Liu, Teizo Yoshimura, Wei Shen, Yingying Le, Scott

More information

Patterns of hemagglutinin evolution and the epidemiology of influenza

Patterns of hemagglutinin evolution and the epidemiology of influenza 2 8 US Annual Mortality Rate All causes Infectious Disease Patterns of hemagglutinin evolution and the epidemiology of influenza DIMACS Working Group on Genetics and Evolution of Pathogens, 25 Nov 3 Deaths

More information

Integration Solutions

Integration Solutions Integration Solutions (1) a) With no active glycosyltransferase of either type, an ii individual would not be able to add any sugars to the O form of the lipopolysaccharide. Thus, the only lipopolysaccharide

More information

Mutation analysis of a Chinese family with oculocutaneous albinism

Mutation analysis of a Chinese family with oculocutaneous albinism /, 2016, Vol. 7, (No. 51), pp: 84981-84988 Mutation analysis of a Chinese family with oculocutaneous albinism Xiong Wang 1, Yaowu Zhu 1, Na Shen 1, Jing Peng 1, Chunyu Wang 1, Haiyi Liu 2, Yanjun Lu 1

More information

Supporting Information

Supporting Information Supporting Information Molecular Recognition Based DNA Nanoassemblies on the Surfaces of Nanosized Exosomes Shuo Wan,, Liqin Zhang,,, Sai Wang, Yuan Liu,, Cuichen Wu, Cheng Cui, Hao Sun, Muling Shi, Ying

More information

Loyer, et al. microrna-21 contributes to NASH Suppl 1/15

Loyer, et al. microrna-21 contributes to NASH Suppl 1/15 Loyer, et al. microrna-21 contributes to NASH Suppl 1/15 SUPPLEMENTARY MATERIAL: Liver MicroRNA-21 is Overexpressed in Non Alcoholic Steatohepatitis and Contributes to the Disease in Experimental Models

More information

Supplementary information

Supplementary information Supplementary information Full methods The conduct of the study was approved by an NHS research ethical committee prior to commencement (reference 12/WS/0288) and was conducted according to the principles

More information

Ho Young Jung Hye Seung Han Hyo Bin Kim 1 Seo Young Oh 1 Sun-Joo Lee 2 Wook Youn Kim

Ho Young Jung Hye Seung Han Hyo Bin Kim 1 Seo Young Oh 1 Sun-Joo Lee 2 Wook Youn Kim Journal of Pathology and Translational Medicine 2016; 50: 138-146 ORIGINAL ARTICLE Comparison of Analytical and Clinical Performance of HPV 9G DNA Chip, PANArray HPV Genotyping Chip, and Hybrid-Capture

More information

L I F E S C I E N C E S

L I F E S C I E N C E S 1a L I F E S C I E N C E S 5 -UUA AUA UUC GAA AGC UGC AUC GAA AAC UGU GAA UCA-3 5 -TTA ATA TTC GAA AGC TGC ATC GAA AAC TGT GAA TCA-3 3 -AAT TAT AAG CTT TCG ACG TAG CTT TTG ACA CTT AGT-5 OCTOBER 31, 2006

More information

Bacterial Gene Finding CMSC 423

Bacterial Gene Finding CMSC 423 Bacterial Gene Finding CMSC 423 Finding Signals in DNA We just have a long string of A, C, G, Ts. How can we find the signals encoded in it? Suppose you encountered a language you didn t know. How would

More information

Isolation and Genetic Characterization of New Reassortant H3N1 Swine Influenza Virus from Pigs in the Midwestern United States

Isolation and Genetic Characterization of New Reassortant H3N1 Swine Influenza Virus from Pigs in the Midwestern United States JOURNAL OF VIROLOGY, May 2006, p. 5092 5096 Vol. 80, No. 10 0022-538X/06/$08.00 0 doi:10.1128/jvi.80.10.5092 5096.2006 Copyright 2006, American Society for Microbiology. All Rights Reserved. Isolation

More information

Supplementary Material Hofko M et al., Detection of carbapenemases by real-time PCR and melt-curve analysis on the BD MAX TM System

Supplementary Material Hofko M et al., Detection of carbapenemases by real-time PCR and melt-curve analysis on the BD MAX TM System Supplementary Material Hofko M et al., Detection of carbapenemases by real-time PCR and melt-curve analysis on the BD MAX TM System Supplementary Material and Methods Characterization of isolates by the

More information

CHAPTER 4 RESULTS. showed that all three replicates had similar growth trends (Figure 4.1) (p<0.05; p=0.0000)

CHAPTER 4 RESULTS. showed that all three replicates had similar growth trends (Figure 4.1) (p<0.05; p=0.0000) CHAPTER 4 RESULTS 4.1 Growth Characterization of C. vulgaris 4.1.1 Optical Density Growth study of Chlorella vulgaris based on optical density at 620 nm (OD 620 ) showed that all three replicates had similar

More information

RESEARCH COMMUNICATION. Genotype Frequencies of Cyclooxygenease 2 (COX2) Rare Polymorphisms for Japanese with and without Colorectal Cancer

RESEARCH COMMUNICATION. Genotype Frequencies of Cyclooxygenease 2 (COX2) Rare Polymorphisms for Japanese with and without Colorectal Cancer COX2 Rare Polymorphisms and Colorectal Cancer RESEARCH COMMUNICATION Genotype Frequencies of Cyclooxygenease 2 (COX2) Rare Polymorphisms for Japanese with and without Colorectal Cancer Nobuyuki Hamajima

More information

PATIENTS AND METHODS. Subjects

PATIENTS AND METHODS. Subjects PATIENTS AND METHODS Subjects Twenty-nine morbidly obese subjects involved in a gastric surgery program were enrolled in the study between October 25 and March 21. Bariatric surgery was performed in patients

More information