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| 1. |
Coldwell, M.J. and Morley, S.J.
(2007)
Specific isoforms of translation initiation factor 4GI show differences in translational activity.
Mol. Cell. Biol.
26
,
8448–8460
.
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Notes:
The authors explored the role of five different eukaryotic initiation factor (eIF) 4GI protein isoforms, which are encoded by alternatively spliced mRNAs, by using short interfering RNAs (siRNAs) to silence the eIF4GI gene. Three eIF4GI siRNA target sequences were evaluated for their ability to reduce eIF4GI mRNA levels in HeLa cells. To quantify the extent of gene silencing, a control plasmid that encodes an eIF4GI/Renilla luciferase fusion mRNA was created using the psiCHECK™-2 Vector. Cotransfection of HeLa cells with the eIF4GI siRNAs and psiCHECK™-2 control plasmid resulted in degradation of the eIF4GI/Renilla luciferase mRNA, leading to reduced Renilla luciferase activity and lower light output. The psiCHECK™-2 Vector encodes the firefly luciferase gene, which allowed normalization of Renilla luciferase expression. Firefly and Renilla luciferase activities were measured using the Dual-Luciferase® Reporter Assay System. Quantitative PCR (qPCR) was used to quantify the silencing of endogenous eIF4GI mRNA splice variants. Prior to qPCR, total RNA was isolated from siRNA-expressing HeLa cells, then reverse transcribed using the ImProm-II™ Reverse Transcription System. qPCR was The pGEM®-T Easy Vector was used in the creation of plasmids encoding siRNA-resistant eIF4GI isoforms, which were transfected into siRNA-expressing HeLa cells to restore eIF4GI function.
(0003778) |
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Products: Dual-Luciferase® Reporter Assay System | ImProm-II™ Reverse Transcription System | pGEM®-T Easy Vector System I | psiCHECK™-2 Vector |
| 2. |
Ghosh, S., Lu, Y., Katz, A., Hu, Y., and Li, R.
(2007)
Tumor suppressor BRCA1 inhibits a breast cancer-associated promoter of the aromatase gene (CYP19) in human adipose stromal cells.
Am. J. Physiol. Endocrinol. Metab.
292
,
E246-E252
.
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Notes:
Obesity-associated elevated estrogen increases the risk for breast cancer in postmenopausal women. The rate limiting step in the synthesis of estrogen from androgen is catalyzed by the aromatase enzyme. Normally this enzyme is expressed under a weak promoter in adipose tissue; however in breast cancer a second, strong ovary-specific promoter (PII) drives expression of aromatase. This study investigated the relationship of BRCA1 and aromatase expression. RNA isolated from BRCA-1 siRNA-treated adipose stromal cells was reverse transcribed using the ImProm-II™ Reverse Transcription System. The authors show that siRNA knockdown of BRCA1 resulted in activation of the PII promoter, suggesting that BRCA1 can modulate estrogen biosynthesis in adipose tissue.
(0003606) |
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Products: ImProm-II™ Reverse Transcriptase | ImProm-II™ Reverse Transcription System |
| 3. |
Witkowski, J.M., Soroczynska-Cybula, M., Bryl, E., Smolenska, Z., and Jozwik, A.
(2007)
Klotho—a Common Link in Physiological and Rheumatoid Arthritis-Related Aging of Human CD4 Lymphocytes
J. Immunology
178
,
771–777
.
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Notes:
Klotho knockout mice exhibit a phenotype of precocious aging, organ failure, osteoporosis. Humans with specific Klotho alleles are at increased risk for osteoporosis, atherosclerosis and decreased lifespan. The authors of this study looked at the expression of Klotho in CD4+ lymphocytes in patients suffering from rheumatoid arthritis and age-matched healthy individuals. cDNA was prepared from total RNA isolated from purified CD4+ lymphocytes using the ImProm-II™ Reverse Transcription System. Klotho expression, protein level and activity was decreased in the lymphocytes from the RA patients.
(0003654) |
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Products: ImProm-II™ Reverse Transcriptase | ImProm-II™ Reverse Transcription System |
| 4. |
Tringali, C., Lupo, B., Anastasia, L., Papini, N., Monti, E., Bresciani, R., Tettamanti, G. and Venerando, B.
(2007)
Expression of sialidase Neu2 in leukemic K562 cells induces apoptosis by impairing Bcr-Abl/Src kinases signaling.
J. Biol. Chem.
282
,
14364–14372
.
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Notes:
The authors transfected the myleoid leukemic cell line K562 with the cytosolic sialidase Neu2. Expression of Neu2 resulted in a significant decrease in mRNA levels for the anti-apoptotic factors Bcl-XL and Bcl-2 as determined by real-time PCR. Reverse transcription was carried out with 1µg of total RNA using the ImProm-II™ Reverse Transcription System and random hexamers. cDNA representing 10ng of total RNA was used in a real-time PCR to quantitate Bcl-XL and Bcl-2 mRNA levels.
(0003725) |
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Products: ImProm-II™ Reverse Transcription System |
| 5. |
Slot, K.A., Voorendt, M., de Boer-Brouwer, M., van Vugt, H.H. and Teerds, K.J.
(2006)
Estrous cycle dependent changes in expression and distribution of Fas, Fas ligand, Bcl-2, Bax, and pro- and active caspase-3 in the rat ovary.
J. Endocrinol.
188
,
179–192
.
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Notes:
The authors examined the locations and levels of apoptosis-related Fas, Fas ligand, Bcl-2, Bax and caspase-3 proteins in ovarian tissue throughout the rat estrus cycle. Protein levels were determined using Western blotting, and proteins were localized by immunhistochemistry. The presence of Fas, Fas ligand, Bcl-2, Bax and caspase-3 mRNA in various ovarian tissues was monitored by RT-PCR. Reverse transcription was performed using the ImProm-II™ Reverse Transcription System, 1µg of RQ1 RNase-free DNase-treated RNA and an oligo(dT) primer. One microliter of the reverse transcription reaction was amplified by PCR, and 10µl of the amplification products were analyzed by agarose gel electrophoresis.
(0003724) |
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Products: ImProm-II™ Reverse Transcription System | RQ1 RNase-Free DNase |
| 6. |
Denyer, M.S., Wileman, T.E., Stirling, C.M.A., Zuber, B., and Takamatsu, H.
(2006)
Perforin expression can define CD8 positive lymphocyte subsets in pigs allowing phenotypic and functional analysis of natural killer, cytotoxic T, natural killer T and MHC un-restricted cytotoxic T-cells.
Vet. Immunol. Immunopathol.
110
,
279-92
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Notes:
In this study, GoTaq® DNA Polymerase was used in two-step RT-PCR. The ImProm-II™ Reverse Transcription System was first used to produce cDNA using an oligo d(T)15 primer. PCR was then performed using GoTaq® DNA Polymerase. Each reaction contained 2μl cDNA, 10μl GoTaq® Reaction Buffer, 1μl dNTP (10mM), 0.2μl GoTaq® DNA Polymerase, 1μl each primer (10pmol) and 34.8μl nuclease-free water. PCR was performed at 94°C for 30 seconds, 55°C for 30 seconds, 72°C for 60 seconds for 35 cycles, and 72°C for 10 minutes.PCR products were visualized by agarose gel electrophoresis containing ethidium bromide and then sequenced.
(0003368) |
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Products: GoTaq® DNA Polymerase | ImProm-II™ Reverse Transcriptase | ImProm-II™ Reverse Transcription System |
| 7. |
Rosenkilde, M.M., Benned-Jensen, T., Andersen, H., Holst, P.J., Kledal, T.N., Luttichau, H.R., Larsen, J.K., Christensen, J.P. and Schwartz, T.W.
(2006)
Molecular pharmacological phenotyping of EBI2. An orphan seven-transmembrane receptor with constitutive activity.
J. Biol. Chem.
281
,
13199-13208
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Notes:
The expression level of the seven-transmembrane Epstein-Barr virus-induced receptor 2 (EBI2) was measured in peripheral blood mononuclear cells. Total RNA was isolated from T-lymphocytes, B-lymphocytes, monocytes and NK cells, and reverse transcribed using the ImProm-II™ Reverse Transcription System. The resulting cDNA was quantitated using real-time PCR.
(0003449) |
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Products: ImProm-II™ Reverse Transcriptase | ImProm-II™ Reverse Transcription System |
| 8. |
Kim, H.J., Ryu, H., Hong, S.H., Woo, H.R., Lim, P.O., Lee, I.C., Sheen, J., Nam, H.G. and Hwang, I.
(2006)
Cytokinin-mediated control of leaf longevity by AHK3 through phosphorylation of ARR2 in Arabidopsis.
Proc. Natl. Acad. Sci. USA
103
,
814-819
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Notes:
The authors characterize an Arabidopsis cytokinin-receptor mutant. RNA was isolated from leaf tissue from wildtype and transgenic Arabidopsis plants expressing various components of the cytokinin signaling pathway. The RNA was reverse transcribed using the ImProm-II™ Reverse Transcription System, and real-time PCR was performed to quantitate response regulator proteins in the signaling pathway.
(0003450) |
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Products: ImProm-II™ Reverse Transcriptase | ImProm-II™ Reverse Transcription System |
| 9. |
Lee, S.J., Jang, B.C., Lee, S.W., Yang, Y.I., Suh, S.I., Park, Y.M., Oh, S., Shin, J.G., Yao, S., Chen, L. and Choi, I.H.
(2006)
Interferon regulatory factor-1 is prerequisite to the constitutive expression and IFN-gamma-induced upregulation of B7-H1 (CD274).
FEBS Lett.
580
,
755-762
.
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Notes:
Many cancer cells upregulate the co-signaling molecule B7-H1, confering resistance to anti-tumor immunity. The ability of interferon regulatory factor-1 (IRF-1) to upregulate B7-H1 expression was characterized by cloning fragments of the B7-H1 promoter upstream of the firefly luciferase reporter gene in the pGL3-Basic Vector and monitoring luciferase expression using the Dual Luciferase® Reporter Assay System. Firefly luciferase measurements were normalized using Renilla luciferase (pRL-CMV Vector). Putative IRF-1 binding sites in the B7-H1 promoter were identified using the Gel Shift Assay System. RT-PCR was used to examine B7-H1 mRNA levels in interferon-γ-treated or untreated A549 cells exposed to various concentrations of IRF-1 siRNA. cDNA synthesis was performed with the ImProm-II™ Reverse Transcription System.
(0003451) |
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Products: Dual-Luciferase® Reporter 1000 Assay System | Dual-Luciferase® Reporter Assay System | Dual-Luciferase® Reporter Assay System 10-Pack | Gel Shift Assay Core System | Gel Shift Assay System | ImProm-II™ Reverse Transcriptase | ImProm-II™ Reverse Transcription System | pGL3-Basic Vector | pRL-CMV Vector |
| 10. |
Grisaru, D., Pick, M., Perry, C., Sklan, E.H., Almog, R., Goldberg, I., Naparstek, E., Lessing, J.B., Soreq, H. and Deutsch, V.
(2006)
Hydrolytic and nonenzymatic functions of acetylcholinesterase comodulate hemopoietic stress responses.
J. Immunol.
176
,
27-35
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Notes:
Expression levels of transcription factors critical for hemopoiesis in bone marrow were determined using real-time quantitative PCR. First, total RNA was isolated from mouse bone marrow, treated with DNase I, then reverse transcribed using the ImProm-II™ Reverse Transcription System. Each reaction included 2.4µl of 25mM MgCl2, 4µl of 5X buffer, 1µl of reverse transcriptase, 1µl of dNTP mix (10mM each), 1µl of 50µM random hexamers, 0.5µl of RNasin® Ribonuclease Inhibitor (20U), and 2µl of sample RNA (200ng/µl).
(0003454) |
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Products: ImProm-II™ Reverse Transcriptase | ImProm-II™ Reverse Transcription System | RNasin® Ribonuclease Inhibitor |
| 11. |
Fett, T., Zecchinon, L., Baise, E., and Desmecht, D.
(2004)
The bovine (Bos taurus) CD11a-encoding cDNA: molecular cloning, characterisation and comparison with the human and murine glycoproteins.
Gene
325
,
97–101
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Notes:
In this paper, ImProm-II™ Reverse Transcriptase was used to generate a full length, ~4560 bp cDNA of bovine CD11 messenger RNA from PMA-stimulated BL-3 bovine B cell lymphoma cells. The full length cDNA was amplified using Invitrogen’s Elongase amplification technology. The amplification products were directly sequenced to yield the full sequence of the cDNA.
(0002848) |
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Products: ImProm-II™ Reverse Transcriptase | ImProm-II™ Reverse Transcription System |
| 12. |
Grubenmann, C.E., Frank, C.G., Hulsmeier, A.J., Schollen, E., Matthijs, G., Mayatepek, E., Berger, E.G., Aebi, M. and Hennet, T.
(2004)
Deficiency of the first mannosylation step in the N-glycosylation pathway causes congenital disorder of glycosylation type Ik.
Hum. Mol. Genet.
13
,
535-542
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Notes:
cDNA was synthesized from 5μg total RNA from patient skin fibroblasts using the specific ALG1 β1,4 mannosyltransferase primer in the presence of 5% DMSO and 1 unit of ImProm-II™ Reverse Transcriptase. Reverse transcription reactions were performed at 42°C for one hour. PCR amplification of the cDNA produced ~1,200bp amplimers.
(0003180) |
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Products: ImProm-II™ Reverse Transcriptase | ImProm-II™ Reverse Transcription System |
| 13. |
van Eden, M.E., Byrd, M.P., Sherrill, K.W. and LLoyd, R.E.
(2004)
Translation of cellular inhibitor of apoptosis protein 1 (c-IAP1) mRNA is IRES mediated and regulated during cell stress.
RNA
10
,
469-481
.
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Notes:
The authors investigate a potential internal ribosome entry site (IRES) in the 5´ untranslated region (UTR) of cellular inhibitor of apoptosis protein 1 (c-IAP1). The c-IAP1 5´ UTR was amplified, cloned into pGEM®-T Vector, sequenced, then inserted into a dicistronic reporter vector between Renilla and firefly luciferase sequences. Using the Dual-Luciferase® Reporter Assay System, IRES activity was evaluated in Rabbit Reticulocyte Lysate and transiently transfected cells. The pSV-β-Galactosidase Control Vector was used as a control for transfection efficiency. Because splicing events were removing part of the Renilla luciferase coding region, the authors chose to use RNA transfection of cells. The ImProm-II™ Reverse Transcription System was used for the reverse transcription step of RT-PCRs to amplify intercistronic regions of the dicistronic RNA to examine mRNA splicing.
(0003429) |
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Products: Dual-Luciferase® Reporter Assay System | Dual-Luciferase® Reporter Assay System 10-Pack | ImProm-II™ Reverse Transcriptase | ImProm-II™ Reverse Transcription System | pSV-β-Galactosidase Control Vector |
| 14. |
Audige, A., Schlaepfer, E., Bonanomi, A., Joller, H., Knuchel, M.C., Weber, M., Nadal, D. and Speck, R.F.
(2004)
HIV-1 does not provoke alteration of cytokine gene expression in lymphoid tissue after acute infection ex vivo.
J. Immunol.
172
,
2687-2696
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Notes:
The authors used real-time quantitative PCR to characterize cytokine response after HIV infection of human lymphoid tissues. To synthesize first-strand cDNA, total RNA was reverse transcribed using the ImProm-II™ Reverse Transcription System: 200U of ImProm-II Reverse Transcriptase, 2µg of total RNA, 500ng of oligo(dT) primer, 500µM dNTPs 3mM MgCl2 and 24U of RNase inhibitor in 1x ImProm-II™ reaction buffer. To obtain viral stocks for infection, 293T cells were transfected with the proviral plasmids pNL4-3 and pYU-2 using the ProFection® Mammalian Transfection System—Calcium Phosphate.
(0003455) |
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Products: ImProm-II™ Reverse Transcriptase | ImProm-II™ Reverse Transcription System | ProFection® Mammalian Transfection System—Calcium Phosphate |
| 15. |
Altmann, S.M., Mellon, M.T., Distel, D.L. and Kim, C.H.
(2003)
Molecular and functional analysis of an interferon gene from the zebrafish, Danio rerio.
J. Virol.
77
,
1992-2002
.
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Notes:
The pGEM®-T Easy Vector was used to subclone products of a 5´ RACE reaction. A promoter construct, assembled in the pGL3 Basic Vector, was co-transfected with a zebrafish interferon expression vector in the ZF4 zebrafish embryo fibroblast cell line using the TransFast™ Reagent (details provided). Luciferase levels were examined with the BrightGlo™ Luciferase Assay Reagent. Induction of zebrafish mRNA was also examined in zebrafish liver cells (ZFL) following treatment with the known interferon inducer, poly(I)-poly(C). RNA was extracted and reverse transcribed using ImProm-II™ Reverse Transcriptase. The resulting cDNA was used for quantitative, real-time RT-PCR with a SYBR green-based assay.
(0002627) |
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Products: Bright-Glo™ Luciferase Assay System | ImProm-II™ Reverse Transcriptase | ImProm-II™ Reverse Transcription System | Luciferase Assay System | pGEM®-T Easy Vector System I | pGEM®-T Easy Vector System II | pGL3-Basic Vector | TransFast™ Transfection Reagent |
| 16. |
Lee, D.K., Geiser, J., Dufner-Beattie, J. and Andrews, G.K.
(2003)
Pancreatic metallothionein-I may play a role in zinc homeostasis during maternal dietary zinc deficiency in mice
J. Nutr.
133
,
45-50
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Notes:
The ImProm-II™ Reverse Transcriptase was used to amplify the protein coding region of a 2,264 base mouse MT-1 gene mRNA and GAPDH from mouse total RNA. RT-PCR was accomplished with Pfu DNA polymerase. The RT-PCR product was used to make a probe for Northern analysis.
(0002609) |
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Products: ImProm-II™ Reverse Transcriptase | ImProm-II™ Reverse Transcription System |
| 17. |
Higashi, K., Inagaki, Y., Suzuki, N., Mitsui, S., Mauviel, A., Kaneko, H. and Nakatsuka, I.
(2003)
Y-box-binding protein YB-1 mediates transcriptional repression of human alpha2(I) collagen gene expression by interferon-gamma.
J. Biol. Chem.
278
,
5156-5162
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Notes:
The Y-box-binding protein YB-1 was examined through the use of a reporter plasmid with wildtype and mutant putative Y-box binding sites. The binding sites were constructed in the pGL3 Basic Vector and measured using the Luciferase Assay System. Studies were performed in normal human dermal fibroblasts. Expression of YB-1 was found to repress expression of the COL1A2 gene at the transcriptional level. Confirmation of this dose-dependent inhibition was through measurement of the steady-state mRNA levels by quantitative, real-time RT-PCR. The reverse transcription portion of the quantitative, real-time RT-PCR reactions were performed with ImProm-II™ Reverse Transcriptase followed by a TaqMan-type quantitative PCR step.
(0002623) |
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Products: ImProm-II™ Reverse Transcriptase | ImProm-II™ Reverse Transcription System | Luciferase Assay System | pGL3-Basic Vector |
| 18. |
Dolci, S., Levati, L., Pellegrini, M., Faraoni, I., Graziani, G., Di Carlo, A. and Geremia, R.
(2003)
Stem cell factor activates telomerase in mouse mitotic spermatogonia and in primordial germ cells.
J. Cell Sci.
115
,
1643-1649
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Notes:
RT-PCR was used to assess the response of mitotic spermatogonia to Kit1. Spermatogonia were cultured with and without Kit1 and RNA harvested after 24 hours. RT-PCR was performed in a two-step reaction using first the ImProm-II™ Reverse Transcriptase in a 20µl reaction. One microliter of the RT reaction was removed and amplified in a 50µl PCR using the PCR Master Mix.
(0002626) |
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Products: ImProm-II™ Reverse Transcriptase | ImProm-II™ Reverse Transcription System | PCR Master Mix |
| 19. |
Dufner-Beattie, J., Wang, F., Kuo, Y.M., Gitschier, J., Eide, D. and Andrews, G.K.
(2003)
The Acrodermatitis enteropathica gene ZIP4 encodes a tissue-specific, zinc-regulated zinc transporter in mice.
J. Biol. Chem.
278
,
33474-33481
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Notes:
Improm-II™ Reverse Transcriptase was used to make cDNAs of mouse ZIP4, a zinc metal ion transporter. The authors used 1μg of total RNA for the reverse transcription reaction. Some reverse transcription reaction products were used to make probes for Northern blot analysis of expression in various tissues. Others were used to make mammalian expression constructs for transient transfection experiments. Details of each application are provided.
(0002722) |
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Products: ImProm-II™ Reverse Transcriptase | ImProm-II™ Reverse Transcription System |
| 20. |
Dominski, Z., Yang, X.C., Purdy, M. and Marzluff, W.F.
(2003)
Cloning and characterization of the Drosophila U7 small nuclear RNA.
Proc. Natl. Acad. Sci. U S A
100
,
9422-9427
.
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Notes:
Improm-II™ Reverse Transcriptase was used to clone U7T snRNA isolated from Drosophila nuclear extracts. The authors used 1ng of extracted U7T snRNA, 30ng of a U7T primer, and 1μl of the Improm-II™ Reverse Transcriptase. cDNAs from the reaction were PCR-amplified and cloned. The resultant clones were used to make probes for Northern blot analysis.
(0002723) |
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Products: ImProm-II™ Reverse Transcriptase | ImProm-II™ Reverse Transcription System |
| 21. |
Tay, E.S.E., Guven, K.L., Subramaniam, N., Polly, P., Issa, L.L., Hardeman, P.W. and Hardeman, E.C.
(2003)
Regulation of alternative splicing of Gtf2ird1and its impact on slow muscle promoter activity.
Biochem. J.
374
,
359–367
.
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Notes:
In this paper, ImProm-II™ Reverse Transcriptase was used to make cDNAs of MusTRD using template RNA isolated from C2C12 myotubes (C2C12) and various mouse hind limb muscle tissues. One microliter of the cDNA products from the reaction was used in real-time PCR analysis using primers for the alternately spliced MusTRD products and SYBR® Green Dye. Data is presented as relative expression compared with the C2C12 cell line or as gel images of the resultant amplimers.
(0002844) |
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Products: ImProm-II™ Reverse Transcriptase | ImProm-II™ Reverse Transcription System |
| 22. |
Tanaka, T., Hanafusa, N., Ingelfinger, J.R., Ohse, T., Fujita, T., and Nangaku, M.
(2003)
Hypoxia induces apoptosis in SV40-immortalized rat proximal tubular cells through the mitochondrial pathways, devoid of HIF1-mediated upregulation of Bax.
Biochem. Biophys. Res. Commun.
309
,
222–231
.
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Notes:
ImProm-II™ Reverse Transcriptase was used in real time RT-PCR to measure the ratio of Bax to Bcl-2 in immortalized rat proximal tubular cells (IRPTCs) cultured under normoxic or hypoxic conditions. The researchers used 1μg of total RNA in the reverse transcription reaction. Qiagen’s QuantiTest CYBR Green PCT Kit was used to quantify PCR products. Promega’s terminal deoxynucleotidyl transferase (TdT) was also used for TdT-mediated dUTP nick end labeling (TUNEL) assays on the cells. The TUNEL-stained cells were analyzed by FACS analysis. Data from these experiments was expressed as percent apoptotic cells.
(0002849) |
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Products: ImProm-II™ Reverse Transcriptase | ImProm-II™ Reverse Transcription System | Terminal Deoxynucleotidyl Transferase, Recombinant |
| 23. |
Guarino, L.A. Mistretta, T.-A. and Dong, W.
(2002)
Baculorus lef-12 is not required for viral replication.
J. Virol.
76
,
12032-12043
.
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Notes:
RT-PCR was used to confirm the expression of the lef-12 gene at various times post-infection. The RT step was performed using ImProm-II™ Reverse Transcriptase in a 20µl reaction volume. One microliter of the RT reaction was amplified in a 50µl PCR amplification reaction using the PCR Master Mix.
(0002628) |
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Products: ImProm-II™ Reverse Transcriptase | ImProm-II™ Reverse Transcription System | PCR Master Mix |