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| 1. |
Liu, Q., Fu, H., Sun, F., Zhang, H., Tie, Y., Zhu, J., Xing, R., Sun, Z. and Zheng, X.
(2008)
miR-16 family induces cell cycle arrest by regulating multiple cell cycle genes.
Nucleic Acids Res.
36
,
5391–404
.
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Notes:
To identify microRNA targets, the authors created a Drosha-knockdown cell line and confirmed depletion of Drosha and three randomly selected miRNAs in these cells by quantitative RT-PCR, using β-actin as a control. The reverse transcription step was performed using the ImProm-II™ Reverse Transcription System. The authors then performed microarray analysis to monitor expression of transcripts to determine which were upregulated as a result of Drosha depletion; cRNA used in these microarray experiments was synthesized using the T7 RiboMAX™ Express Large Scale RNA Production System. Cyclin D1 was identified as a potential miRNA target. To screen miRNAs that regulate cyclin D1, the authors cloned the cyclin D1 3´ untranslated region downstream of the firefly luciferase gene of the pGL3-Control Vector and measured luciferase levels in transfected cells using the Dual Luciferase Reporter Assay System. Renilla luciferase in the pRL-TK Vector was used as a normalization control.
(0003894) |
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Products: Dual-Luciferase® Reporter Assay System | ImProm-II™ Reverse Transcriptase | pGL3-Control Vector | pRL-TK Vector | T7 RiboMAX™ Express Large Scale RNA Production System |
| 2. |
Adams, B.D., Furneaux, H. and White, B.
(2007)
The micro-RNA miR-206 targets the human estrogen receptor-α, and represses ERα mRNA and protein expression in breast cancer cells.
Mol. Endocrinol.
Mar. 13
,
Epub (ahead of print)
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Notes:
This study investigated the mechanism of silencing of the estrogen receptor α mRNA in the human breast cancer cell line, MCF-7. The authors initially used software for miRNA target prediction to analyze the 3´ UTR of the human ERα gene for potential miR-206 target sites. Two potential targets, designated hERα1 and hERα2, were identified. ERα levels were repressed in a dose-dependent manner in MCF-7 cells transfected with a synthetic pre-miR-206 duplex, and transfection of an miR-206 expression construct into MCF-7 cells also resulted in specific inhibition of ERα expression, as measured by real-time PCR and Northern blot assays. A luciferase reporter assay was then used to determine whether miR-206 interacted directly with the hERα1 and hERα2 sites in the ERα 3´UTR. Luciferase reporter constructs containing either the hERα1 or hERα2 cloned 3´ of the firefly luciferase gene showed miR-206-medisted repression of luciferase expression in HeLa cells. Mutation of the hERα1 or hERα2 sites to disrupt hybridization with the 5´ region of miR-206 restored luciferase activity, as did co-transfection with an miRNA antagonist of miR-206. Transformation of the luciferase constructs into the breast cancer cell lines, MCF-7 and MDA-MB-231, both of which expressed high levels of miR-206 as measured by real-time PCR, resulted in repression of luciferase activity. Treatment with estrogen was then shown to reduce miR-206 levels in MCF-7 cells. Luciferase assays were used to confirm this result, and levels of luciferase activity from the reporter constructs were increased upon exposure to estrogen, indicating that ERα agonists were able to decrease miR-206 levels in MCF- cells.
(0003603) |
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Products: Luciferase Assay System |
| 3. |
Tsuchiya, Y., Nakajima, M., Takagi, S., Taniya, T., and Yokoi, T.
(2006)
MicroRNA regulates the expression of human cytochrome P450 1B1.
Cancer Res.
66
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9090-9098
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Notes:
These authors identified a region complementary to the microRNA miR-27b in the 3´ UTR of the cytochrome p450 CYP1B1 mRNA, and showed that miR-27b was involved in regulation of CYP1B1 expression. The 3´ UTR containing the miRNA target site was cloned downstream of the luciferase gene in the pGL3 Promoter Vector and cotransfected into the miR-27b-positive breast cancer cell line MCF-7 and into miR-27b-negative Jurkat cells. Luciferase expression levels from the reporter vector containing the CYP1B1 3´ UTR sequence were reduced in miR-27b-positive cells, but not in the Jurkat cell controls. Delivery of an antisense oligoribonucleotide directed against miR-27b to MCF-7 cells containing the reporter construct resulted in restoration of luciferase activity. The effects of inhibition of miR-27b on protein levels and enzymatic activity of CYP1B1 were then investigated in MCF-7 cells. CYP1B1 protein levels and enzymatic activity increased significantly in cells transfected with the antisense oligo; the enzymatic activity was measured using a p450-Glo™ Assay. The coding region and 3´ UTR of the CYP1B1 gene were also PCR-amplified, subcloned the into the pTargeT™ Mammalian Expression Vector, and transfected into HEK293 cells. The effect of overexpression of miR-27b on protein levels and enzymatic activity of CYP1B1 was then evaluated in these cells.
(0003622) |
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Products: Dual-Luciferase® Reporter Assay System | Dual-Luciferase® Reporter Assay System 10-Pack | P450-Glo™ CYP1B1 Assay | pGL3-Promoter Vector | pTARGET™ Mammalian Expression Vector System |
| 4. |
Lee, H-J, Palkovits, M. and Young, W.S.
(2006)
miR-7b, a microRNA up-regulated in the hypothalamus after chronic hyperosmolar stimulation, inhibits Fos translation.
Proc. Natl. Acad. Sci. U S A
103
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15669–15674
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Notes:
In this study, the psiCHECK™ Vector was used in an investigation of the interaction between a microRNA and its potential target mRNA. Increased Fos expression in the paraventricular (PVN) and supraoptic (SON) nuclei is associated with hyperosmolality. In an effort to identify microRNAs that may regulate Fos expression, miRNA was isolated from the PVN and SON of mice after 10 days of 2% saline ingestion, and differentially expressed miRNAs were identified by microarray analysis. The miR-7b miRNA, which was overexpressed after saline treatment, was selected for further analysis as the fos gene 3´ UTR contains putative miR-7b binding sites. To investigate the potential interaction between miR-7b and Fos, the 3´ UTR of fos was subcloned downstream of the Renilla luciferase gene in the psiCHECK™ Vector. 293T cells were then co-transfected with the psiCHECK-Fos vector construct (0.8µg) and a vector expressing miR-7b and GFP (2.4µg). Luciferase assays were performed 42 hours post-transfection using the Dual-Luciferase® Reporter Assay System. A 40% reduction in Renilla expression was observed in cells co-transfected with the miR-7b vector compared with cells transfected with psiCHECK-Fos and a control miRNA.
(0003560) |
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Products: Dual-Luciferase® Reporter Assay System | Dual-Luciferase® Reporter Assay System 10-Pack | psiCHECK™-1 Vector | psiCHECK™-2 Vector |
| 5. |
Wang, B., Love, T.M., Call, M.E., Doench, J.G. and Novina, C.D.
(2006)
Recapitulation of short RNA-directed translational gene silencing in vitro.
Mol. Cell
22
,
553-560
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Notes:
microRNAs (miRNAs) are a class of endogenous short RNAs that repress gene expression. To assess miRNA-directed translational silencing, in vitro reactions were performed. Translational gene silencing by miRNAs in vitro requires target mRNAs to possess a 7-methyl G cap and a poly(A) tail. In vitro transcription was performed with 5μg of linearized plasmids containing zero, two, four, or six miCXCR4 binding sites, one siCXCR4 binding site or Renilla luciferase (pRL-TK; control mRNA) using the RiboMax™ T7 Large-Scale RNA Production kit. The mRNAs were modified by 7-methyl-G capping and polyadenylation. Translation was performed using nuclease-treated rabbit reticulocyte lysate containing 0.025pmole of mRNA with miCXCR4 or siCXCR4 binding sites, 0.025pmole Renilla control RNA, and different ratios of CXCR4 siRNA. Reaction products were separated by SDS-PAGE and visualized and quantitated by PhosphorImager analysis.
(0003412) |
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Products: pRL-TK Vector | Rabbit Reticulocyte Lysate System, Nuclease Treated | RiboMAX™ Large Scale RNA Production System—SP6 | RiboMAX™ Large Scale RNA Production System—T7 |
| 6. |
Vinther, J., Hedegaard, M.M., Gardner, P.P., Andersen, J.S. and Arctander, P.
(2006)
Identification of miRNA targets with stable isotope labeling by amino acids in cell culture.
Nucleic Acids Res.
34
,
e107
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Notes:
These authors used stable-isotope labeling of amino acids in culture (SILAC), a method that allows quantitation of relative protein abundance between populations, to investigate the effect of the microRNA miRNA-1 on the HeLa cell proteome. HeLa cells grown in the presence of labeled arginine and lysine were transfected with miRNA-1, and the labeled proteins compared to those from control cells treated with the transfection reagent alone. A set of 16 proteins repressed by miRNA-1 was identified. The 3´ UTR's from 11 of the miRNA-1-regulated genes were tested in a reporter assay, and 6 were shown to repress expression in an miRNA-1-dependent fashion. For the reporter assays, the HSV TK promoter was amplified from the pRL-TK Vector and subcloned into the pGL4.12(luc2CP) Vector, creating the pGL4.12-TK vector. The 3´ UTR regions from suspected target genes were then amplified and subcloned into the pGL4.12-TK construct. The various pGL4.12-TK constructs (0.9µg) were then co-transfected along with pRL-TK (0.1µg) and miRNA-1 (50pmol) into HeLa cells (80,000 cells/well in 12-well plates). Twenty-two hours post-transfection, firefly and Renilla luciferase activities were measured using the Dual-Luciferase® Reporter Assay System.
(0003561) |
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Products: Dual-Luciferase® Reporter Assay System | Dual-Luciferase® Reporter Assay System 10-Pack | pGL4.12[luc2CP] Vector | pRL-TK Vector |
| 7. |
Leaman, D., Chen, P.Y., Fak, J., Yalcin, A., Pearce, M., Unnerstall, U., Marks, D.S., Sander, C., Tuschl, T. and Gaul, U.
(2005)
Antisense-mediated depletion reveals essential and specific functions of microRNAs in Drosophila development.
Cell
121
,
1097–108
.
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Notes:
To monitor the effects of targeted degradation of micro RNA (miRNA) on Drosophila embryos, the authors cloned the full-length 3'UTRs of the proapoptotic factors hid and reaper into the psiCHECK™-2 Vector. The constructs were injected as plasmids (1µg/ml) mixed with 400µM sense and antisense miR-2 2'O-methyl oligoribonucleotides in early embryos. The total volume injected was equal to 5% of egg volume. After 10 hours development, the embryos were washed and lysed under agitation using 60µl lysis buffer and shaken at 750 rpm at room temperature for 30 minutes. The resulting lysate was cleared by centrifugation and three aliquots were tested using the Dual-Luciferase® Reporter Assay System. The Renilla-to-firefly luciferase ratios from three to five independent replicates were averaged and normalized to the value of the miR-6 sense control, the most severe apoptotic phenotype when targeted for depletion. Statistical significance was assessed using the t test.
(0003292) |
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Products: Dual-Luciferase® Reporter 1000 Assay System | Dual-Luciferase® Reporter Assay System | Dual-Luciferase® Reporter Assay System 10-Pack | psiCHECK™-2 Vector |