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Citations Search

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Am. J. Hum. Genet. 97, 555-66. Biallelic mutations in nuclear pore complex subunit NUP107 cause early-childhood-onset steroid-resistant nephrotic syndrome. 2015

Niyake, N., Tsukaguchi, H., Koshimizu, E., Shono, A., Matsunaga, S., Shiina, M., Mimura, Y., Imamura, S., Hirose, T., Okudela, K., Nozu, K., Akioka, Y., Hattori, M., Yoshikawa, N., Kitamura, A., Cheong, H.I., Kagami, S., Yamashita, M., Fugita, A., Miyatake, S., Tsurusaki, Y., Nakashima, M., Saitsu, H., Ohashi, K., Imamoto, N. and Ryo, A.

Notes: The interaction of NUP107 and NUP133 were investigated by several means. First, in vitro expressed, biotinylated NUP107 was mixed with FLAG-tagged NUP133 and pulled down with Streptavidin MagneSphere® Particles and blotted for reaction with Anti-FLAG antibodies. HeLa cells were transfected with a GFP-NUP107 fusion vector using ViaFect™ Transfection Reagent (no details provided) and immunoprecipitated then blotted to identify NUP133 in the pull down. Subcellular localization of GFP-NUP107 was examined as well. RT-PCR analysis of nup107 splicing variants in zebrafish relied on M-MLV Reverse Transcriptase for cDNA synthesis. (4675)

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J. Proteome Res. 13, 5041–50. Large-scale label-free comparative proteomics analysis of polo-like kinase 1 inhibition via the small-molecule inhibitor BI 6727 (Volasertib) in BRAFV600E mutant melanoma cells. 2014

Cholewa, B.D., Pellitteri-Hahn, M.C., Scarlett, C.O. and Ahmad, N.

Notes: Cell pellets of A375 human melanoma cells were lysed by passing through a needle, centrifuged to remove debris and protein quantitated. Twenty micrograms of protein from control and treated lysates were digested with 1µg of Sequencing Grade Modified Trypsin and used for mass spectrometry analysis. A375 cells (5 × 105) were grown in a 10cm dish and treated for 24 hours before isolating RNA and DNA synthesized using M-MLV Reverse Transcriptase. The cDNA was then used in qPCR. A375 cells were plated at 3 × 103 in 96-well half-volume white-wall plates, grown and treated for 24 hours. NAD, NADH and NADPH were determined using the NAD(P)H-Glo™ Detection System or NAD/NADH-Glo™ Assay and luminescence measured. (4963)

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J. Biol. Chem. 282, 9883–94. Cell confluence-induced activation of signal transducer and activator of transcription-3 (Stat3) triggers epithelial dome formation via augmentation of sodium hydrogen exchanger-3 (NHE3) expression. 2007

Su, H.W., Yeh, H.H., Wang, S.W., Shen, M.R., Chen, T.L., Kiela, P.R., Ghishan, F.K. and Tang, M.J

Notes: The authors tested their hypothesis that Na+-H+ exchangers (NHE) are involved in the formation of multicullar dome structures in confluent Madin-Darby canine kidney (MDCK) cells and that the Stat3 pathway is involved in regulation of NHEs. The authors performed semi-quantitative RT-PCR to monitor NHE3 mRNA levels in MDCK cells expressing a constitutive Stat3 mutant or a dominant-negative Stat3 mutant. The reverse transcription step was performed using Promega M-MLV Reverse Transcriptase. RAlso, Stat3 activities in low-density cultures and high-density cultures were compared using a reporter gene assay. Four copies of the Stat3-binding site were cloned upstream of a firefly luciferase reporter gene, and the resulting vector, along with the pRL-TK Vector for normalization, were transfected into MDCK cells. Luciferase activities were measured using the Dual-Luciferase Reporter Assay System. (3910)

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J. Biol. Chem. 282, 21818–21828. Degradation of hsp70 and other mRNAs in Drosophila via the 5´ 3´ pathway and its regulation by heat shock. 2007

Bönisch, C., Temme, C., Moritz, B. and Wahle, E.

Notes: The authors studied hsp70 mRNA degradation in Drosophila Schneider cells. mRNA deadenylation and decay were monitored by Northern blot. Two of the Northern blot probes used to visualize the mRNA decay products were synthesized by transcription of linearized plasmids using T7 RNA Polymerase and [α-32P] UTP. A population of deadenylated mRNA was created by hybridizing mRNA with oligo(dT) and treating with RNase H. CCR4•NOT was identified as the main deadenylase involved in mRNA decay, and the PAN2:PAN3 deadenylase was a minor contributor. RNA interference was used to knock down expression of PAN2 and CAF1, a subunit of CCR4•NOT, to assess the effect on mRNA decay. Reduced expression levels of PAN2 and CAF1 were confirmed by semi-quantitative RT-PCR and Western blotting, respectively. RT-PCR was performed using 1.5µg total RNA and 150 units of MMLV Reverse Transcriptase in a 25µl reaction. One microliter of the RT reaction was used as a template in an 80µl PCR using 0.5 units of GoTaq® DNA Polymerase, 1.5mM MgCl2 and 1X Green GoTaq® Flexi Reaction Buffer. (3707)

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FASEB J. 21, 1893–1901. Low expression of COX-2, reduced cumulus expansion, and impaired ovulation in SULT1E1-deficient mice. 2007

Gershon, E., Hourvitz, A., Reikhav, S., Maman, E. and Dekel, N.

Notes: The authors investigated the role of estrogen inactivation by the SULT1E1-encoded estrogen sulfotransferase in ovulation in mice. Semiquantitative RT-PCR was used to characterize the temporal and tissue-specific expression of SULT1E1 mRNA in ovulating mice. First-strand cDNA synthesis was performed at 37°C for 2 hours using 7.5µg of total RNA, 1µl (0.5µg) of oligo(dT)15, 40 units of RNasin® Ribonuclease Inhibitor and 200 units of M-MLV Reverse Transcriptase. (3913)

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J. Endocrinol. 197, 201–12. Neonatal exposure to bisphenol A modifies the abundance of estrogen receptor alpha transcripts with alternative 5'-untranslated regions in the female rat preoptic area. 2007

Monje, L., Varayoud, J., Luque, E.H. and Ramos, J.G.

Notes: The authors investigated the effect of neonatal bisphenol A (BPA) exposure in rats on expression of estrogen receptor α (ERα) transcripts. Alternative ERα transcripts in preoptic area of treated and untreated rats were quantified using real-time RT-PCR. Reverse transcription was performed using 4µg of total RNA, 200pmol random primers and 300 units M-MLV Reverse Transcriptase. Real-time PCR was performed using SYBR® Green I to quantify amplified products. To determine if the changes in BPA-induced ERα transcript expression were caused by DNA methylation, the methylation status of the five ERα promoters was examined by bisulfite modification. Genomic DNA was isolated from rat tissue using the Wizard® Genomic DNA Purification Kit, denatured with NaOH, then treated with hydroquinone and sodium bisulfite. Prior to methylation-specific PCR, DNA was cleaned up using the Wizard® DNA Purification Resin as directed by the manufacturer. PCR products were cleaned up again using the Wizard® SV Gel and PCR Clean-Up System, then subjected to restriction enzyme digestion and agarose gel electrophoresis to reveal methylation-dependent sequence differences. (3911)

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Mol. Cell. Endocrinol. 264, 50-60. Novel estrogen receptor beta transcript variants identified in human breast cancer cells affect cell growth and apoptosis of COS-1 cells. 2007

Treeck, O., Pfeiler ,G., Horn, F., Federhofer, B., Houlihan, H., Vollmer, A., and Ortmann, O.

Notes: This study identified two novel transcript variants of the estrogen receptor ERβ that were expressed in the ERα-negative breast cancer cell line MDA-MD-231. These variants were identified after amplification of ERβ transcripts from the breast cancer cell line by RT-PCR. The amplification products were then excised from gels and subcloned into the pTARGET™ Mammalian Expression Vector prior to sequencing. COS1 cells, which do not express the estrogen receptor, were then stably transfected with full-length ERβ or one of the splice variants and the effects on cell proliferation, apoptosis, and estrogen response were evaluated. In COS1 cells expressing either ERβ or the transcript variants cell proliferation decreased and basal apoptosis (caspase 3/7 activity) increased, compared to cells transfected with vector alone. Exposure to therapeutic doses of tamoxifen induced apoptosis in cells expressing the full-length ERβ but not in cells expressing either of the variant isoforms. (3618)

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Genetics 173, 2143–2149. Functional Implication of an ARG307GLY Substitution in Corticosteroid Binding Globulin, a Candidate Gene for a QTL Associated with Cortisol Variability and Obesity. 2006

Guyonnet-Duperat, V., Geverink, N., Plastow, G.S., Evans, G., Ousova, O., Croisetiere, C., Foury, A., Richard, E., Mormede, P. and Moisan, M.P.

Notes: In this study, the effects of amino acid substitutions in porcine corticosteroid-binding globulin gene (Cbg) were tested on CBG binding and affinity. Genomic DNA was isolated from whole blood of 92 female pigs studied. Cbg cDNA was obtained by reverse transcribing pig liver total RNA using M-MLV Reverse Transcriptase followed by PCR. The 1257pb cDNA PCR product was ligated into the pTARGET™ Mammalian Expression Vector. The GeneEditor™ in vitro Site-Directed Mutagenesis System was used to introduce four different codon substitutions in the Cbg cDNA. Once created, the mutated and unmodified Cbg cDNA constructs were transfected into HEK-293T (human embryonic kidney) cells. After 48 hours, the supernatant was collected to analyze secreted CBG. (3498)

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Stem Cells 24, 781-792. Human umbilical cord matrix stem cells: Preliminary characterization and effect of transplantation in a rodent model of Parkinson's disease. 2006

Weiss, M.L., Medicetty, S., Bledsoe, A.R., Rachakatla, R.S., Choi, M., Merchav, S., Luo, Y., Rao, M.S., Velagaleti, G. and Troyer, D.

Notes: The gene expression profile of human umbilical cord matrix stem (UCMS) cells was determined by microarray analysis and confirmed by RT-PCR. The 50 most highly expressed genes in UCMS cells tended to fall into several categories: genes associated with neurotrophic effects and morphogenesis, the three germ layers, the undifferentiated state of embryonic stem cells and extracellular adhesion molecules. The biotin dUTP-labeled cDNA probes used to query the UCMS cell microarray were synthesized using 200 units of M-MLV Reverse Transcriptase and 4µg of total RNA. (3469)

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Clin. Chem. 52, 634-642. Quantification of mRNA in whole blood by assessing recovery of RNA and efficiency of cDNA synthesis. 2006

Mitsuhashi, M., Tomozawa, S., Endo, K. and Shinagawa, A.

Notes: The authors developed a method to reverse transcribe poly(A)+ RNA from leukocytes without using oligo(dT) immobilized on a 96-well plate. Four RNA targets, as well as a synthetic control RNA, were reverse transcribed using MMLV Reverse Transcriptase (1X reverse transcription buffer [50mM KCl, 10mM Tris-HCl (pH 8.3), 5.5mM MgCl2, 1nL/µL Tween 20], 1.25 mM each dNTP and 4 units of Recombinant RNasin® Ribonuclease Inhibitor), then quantitated by real-time quantitative PCR. The authors varied the concentration of MMLV Reverse Transcriptase, incubation time, and primer/template ratio] to obtain the maximum yield. Small quantities of MMLV Reverse Transcriptase were sufficient to reverse transcribe short synthetic RNA and abundant RNAs, but approximately 100 units was required for other RNAs. The synthetic control RNA was synthesized using the T7 RiboMAX™ Express Large Scale RNA Production System. (3456)

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Clin. Exp. Immunol. 131, 144–51. Contribution of Vα24+Vβ11+ natural killer T cells in Wilsonian hepatitis. 2005

Kinebuchi, M., Matsuura, A., Ohya, K., Abo, W. and Kitazawa, J.

Notes: To prepare single rat NK cells for reverse transcription, the cells were sorted by flow cytometry and lysed using 0.5% NP-40, 2.2µl of 5X M-MLV Reverse Transcriptase Buffer, 0.3µl of 0.1mol/l DTT, 5.5µl DEPC-treated water and 1U of RNasin® Plus RNase Inhibitor. After a 30-minute incubation on ice, the lysates were heated to 65°C for 90 seconds, cooled to 22°C for 3 minutes and placed back on ice. For the RT reaction, 1U RNasin® Plus RNase Inhibitor, 60 U of M-MLV Reverse Transcriptase, 2.5mmol/l dNTPs and 100µM primers were added to these single-cell lysates. (3390)

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Plant Physiol. 126, 1416–1429. Efficient prenylation by a plant geranylgeranyltransferase-I requires a functional CaaL box motif and a proximal polybasic domain. 2004

Caldelari, D., Sternberg, H., Rodríguez-Concepción, M., Gruissem, W. and Yalovsky, S.

Notes: Using the SV Total RNA Isolation System before RT-PCR, expression levels of AtGGT-IB were compared in flowers, leaves, stems, and root tissues of wildtype and era1-2 Arabidopsis plants. First-strand synthesis was performed with M-MLV Reverse Transcriptase using 500ng of RNA and an equal amount of oligo(dT). A 1:10 dilution of this product was used for amplification. (2855)

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Oncogene 23, 8920-30. Fibronectin and type IV collagen activate ERα AF-1 by c-Src pathway: effect on breast cancer cell motility. 2004

Sisci, D., Aquila, S., Middea, E., Gentile, M., Maggiolini, M., Mastroiami, F., Montanaro, D. and Ando, S.

Notes: MCF-7 cells were incubated for 24 hours in PRF-SFM and then detached and plated on uncoated dishes or dishes coated with 2μg/cm2 poly-L-Lysine (P-Lys) in PBS, 30μg/ml fibronectin (Fn) in PBS or 30μg/ml type IV Collagen (Col) in 10mM acetic acid. The cells plated on uncoated dishes were treated both with and without 10nM estradiol (E2). After 24 hours, total cellular RNA was extracted and reverse transcribed using M-MLV reverse transcriptase. Briefly, reverse transcription was performed on 1μg of total RNA in a final volume of 10μl by incubation at 37°C for 30 min with 200U of M-MLV reverse transcriptase, 0.4μg oligo-dT, 0.5μM dNTP and 24U RNasin® Ribonuclease Inhibitor, followed by heat denaturation for 5 minutes at 95°C. Subsequent PCR analysis was performed on 1μl of the RT product in a final volume of 25μl. Primers were used to amplify the 210bp fragment of PS2 and the 304 bp fragment of cathepsin D. Amplification of 408bp of ribosomal RNA 36B4 was performed as control. The PCR mixture consisted of 1.25U GoTaq® DNA Polymerase, 1X PCR buffer (10mM Tris-HCl, 50mM KCl), 2.5mM MgCl2, 0.2mM each dNTP, 0.6μM of each PS2 primer or cathepsin D primer and 0.2μM of each 36B4 primer. PCR was performed for 20 cycles at 95°C for 1 minute, 59°C for 2 minutes and 72°C for 1 minute. Ten microliters of the PCR products were separated on a 1.2% agarose gel. (3377)

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Oncogene 23(45), 7517-7526. Nuclear insulin receptor substrate 1 interacts with estrogen receptor alpha at ERE promoters. 2004

Morelli, C., Garofalo, C., Sisci, D., del Rincon, S., Cascio, S., Tu, X., Vecchione, A., Sauter, E.R., Miller, W.H. Jr. and Surmacz, E.

Notes: COS-7 cells were harvested and total RNA isolated 24 hours after transfection with plasmids expressing trans-activating proteins. Five micrograms of RNA was reverse transcribed with oligo(dT) primer using the M-MLV Reverse transcriptase. One tenth of the reaction volume (2μl) was then used as template in PCR to amplify a gene presumed to be controlled by the trans-activators (pS2; 210bp) or a control gene (36B4 ribosomal phosphoprotein; 408bp). The PCR products were amplified using GoTaq® DNA polymerase. (3203)

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Clin. Can. Res. 9, 5874–5879. Sensitization to the cytotoxicity of cisplatin by transfection with nucleotide excision repair gene Xeroderma Pigmentosun Group A antisense RNA in human lung adenocarcinoma cells. 2003

Wu, X., Fan, W., Xu, S., and Zhou, Y.

Notes: The pGL3-Promoter Vector was treated with various concentrations of the chemotherapeutic cancer and DNA cross-linking agent, cisplatin. The damaged pGL3-Promoter Vector and the pSV-β-Galactosidase Control Vector were then transiently transfected into the human lung adenocarcinoma cell line, A549, and later analyzed for luciferase activity using the Luciferase Assay System with Reporter Lysis Buffer. The researchers also used M-MLV Reverse Transcriptase to clone the antisense sequence to XPA (Xeroderma Pigmentosum group A) mRNA.  The cloned antisense sequence was then expressed in A549 cells for its effect on XPA gene expression. (2833)

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Proc. Natl. Acad. Sci. USA 100, 13773-13778. Shade is the Drosophila P450 enzyme that mediates the hydroxylation of ecdysone to the steroid insect molting hormone 20-hydroxyecdysone. 2003

Petryk, A., Warren, J.T., Marqués, G., Jarcho, M.P., Gilbert, L.I., Kahler, J., Parvy, J.P., Li, Y., Dauphin-Villemant, C. and O'Connor, M.B.,

Notes: Researchers used the SV Total RNA Isolation System to isolate total RNA from various tissues from Drosophila larvae. The isolated RNA from various tissues was used in reverse transcription reactions (using M-MLV Reverse Transcriptase) to make cDNAs of the shade (Shd) gene message. PCR amplification of Shd cDNA demonstrated that Shd was specifically expressed in certain Drosophila tissues. (2784)

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Invest. Ophthalmol. Vis. Sci. 43, 72-81. Differential effect of activin A and BMP-7 on myofibroblast differentiation and the role of the Smad signaling pathway. 2002

You, L. and Kruse, F.E.

Notes: Total RNA was isolated from human corneal tissue with the RNAgents® Total RNA Isolation System.  The total RNA was further processed into the poly(A)+ fraction through the use of the PolyATtract® mRNA Isolation System. The isolated mRNA was used for RT-PCR and Northern analysis. RT-PCR was performed using M-MLV Reverse Transcriptase. (2569)

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J. Clin. Invest. 109, 923-930. The melanin-concentrating hormone receptor 1, a novel target of autoantibody responses in vitiligo 2002

Kemp, E.H., Waterman, E.A., Hawes, B.E., O'Neill, K., Gottumukkala, R.V.S.R.K., Gawkrodger, D.J., Weetman, A.P., and Watson, P.F.

Notes: T4 Polynucleotide Kinase was used to phosphorylate primers before ligating them into a phagemid vector to allow cloning of Sfi I-restricted DNA fragments. Phagemid was prepared from XL1-Blue cells using the Wizard® Minipreps DNA Purification System. Inserts were PCR amplified from the phagemid and purified using the Wizard® PCR Preps DNA Purification System. In separate experiments, the cDNA for the melanin concentrating hormone receptor 1 cDNA was cloned from total melanocyte RNA using MMLV Reverse Transcriptase. In vitro translation of the cDNA was performed using the TnT T7 Coupled Reticulocyte Lysate System and Canine Pancreatic Microsomal Membranes. (2602)

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Clin. Can. Res. 8, 2085-2090. The tumor suppressor gene LKB1 is associated with prognosis in human breast carcinoma 2002

Shen, Z., Wen, X-F., Lan, F., Shen, Z-Z., Shao, Z-M.

Notes: MMLV reverse transcriptase and RNasin Ribonuclease Inhibitor were used to produce cDNA from total RNA isolated from transfected and untransfected human breast cancer cells. The coding region of LKB1 was amplified by PCR using Taq DNA Polymerase and buffer from Promega. (2601)

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J. Biol. Chem. 277, 12023-12031. Transcriptional regulation of Kaposi's sarcoma-associated herpesvirus-encoded oncogene viral interferon regulatory factor by a novel transcriptional silencer, Tis. 2002

Wang, X.-P., Zhang, Y.-J., Deng, J.-H., Pan, H.-Y., Zhou, F.-C., Gao, S.-J.

Notes: Total herpesviral RNA was isolated from HeLa cells, BC-1 cells, 293 cells and human heart tissue using the RNAgents® Total RNA Isolation System. The isolated RNA was used for RT-PCR.  First-strand cDNA synthesis was accomplished using Promega's M-MLV Reverse Transcriptase and Random Hexamer Primers. (2563)

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Physiol. Genomics 5, 187–192. Microarray analysis of nicotine-induced changes in gene expression in endothelial cells. 2001

Zhang, S., Day, I.N.M. and Ye, S.

Notes: The SV Total RNA Isolation System was used to isolate total RNA from primary human coronary artery endothelial cells. The isolated RNA was used to make gene array 33P-labeled targets on nylon cDNA microarray filters using Oligo(dT) and M-MLV Reverse Transcriptase, RNase H-. The same material was used for first-strand synthesis in RT-PCR. (2697)

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J. Bacteriol. 183, 7094-7101. Quantification of expression of Staphylococcus epidermidis housekeeping genes with Taqman quantitative PCR during in vitro growth and under different conditions. 2001

Vandecasteele, S.J., Peetermans, W.E., Merckx, R. and Van Eldere, J.

Notes: M-MLV Reverse Transcriptase and RNasin® Ribonuclease Inhibitor were used in a reaction to generate first strand cDNA.  The generated cDNA was used as a template for quantitative PCR in a Taqman® Assay.  The pGEM®-T Vector System was used to clone copies of each target gene so that standards could be generated.  The Wizard® Genomic DNA Purification Kit was used to isolated genomic DNA from the S. epidermidis to generate a standard curve for quantitation of genomic DNA contamination of samples. (2291)

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J. Bacteriol. 183, 6801–6806. Transcriptional Regulation of furA and katG upon Oxidative Stress in Mycobacterium smegmatis. 2001

Milano, A., Forti, F., Sala, C., Riccardi, G. and Ghisotti, D.

Notes: Total RNA was isolated from Mycobacterium smegmatis for Northern blot analysis and RT-PCR. Transcripts for furA and katG were amplified by RT-PCR using Promega's MMLV Reverse Transcriptase and cloned into the pGEM®-T Easy vector. Northern blot probes for furA and katG were synthesized by in vitro transcription from the pGEM®-T Easy vector. (2310)

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J. Immunol. 161, 474-480. System administration of endotoxin induces bronchopulmonary hyperactivity dissociated from TNF-alpha formation and neutrophil sequestration into the murine lungs. 1998

Lefort, J., Singer, M., Leduc, D., Renesto, P., Nahori, M.A., Huerre, M., Créminon, Chignard, M., Vargaftig, B.B.

Notes: Poly A+ RNA was isolated directly from PBS-perfused mouse lungs with the PolyATtract® System 1000. The isolate poly A+ RNA was used for quantitative two-step RT-PCR using M-MLV RNase Hminus Reverse Transcriptase as well as RNasin® Ribonuclease inhibitor for first step cDNA synthesis. (0821)

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Oncogene 16(5), 643-654. The C-terminus of the HTLV-1 Tax oncoprotein mediates interaction with the PDZ domain of cellular proteins. 1998

Rousset, R., Fabre, S., Desbois, C., Bantignies, F. and Jalinot, P.

Notes: The system was used to isolate total RNA from Jurkat cells. The isolated RNA was used for RT-PCR. The RT reaction was catalyzed by MMLV Reverse Transcriptase. (1662)

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