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View additional product information for mirVana™ miRNA Inhibitor, Negative Control #1 - FAQs (4464076, 4464078, 4464077, 4464079)
16 product FAQs found
Using TaqMan miRNA assays, you can measure the level of miRNA of interest. If the miRNA is bound to the synthetic anti-miRs and/or mirVana inhibitors transfected, the duplex should not be detected, indicating efficient miRNA inactivation. Please note, this only works when total RNA sample isolated is not heat denatured when RT primer is added, as heat denaturation will result in complex dissociation and subsequent detection of microRNA by TaqMan miRNA assay even when cells have been transfected properly. Please see the protocol for the TaqMan MicroRNA Cells-to-CT kit (Cat. No. 4391848) for more details.
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Mimics and inhibitors can be transfected or electroporated into cells using parameters similar to those used for siRNA.
Using mirVana miRNA Mimics- For the following applications, use a cell line that expresses relatively low levels of the mature miRNA of interest.
- Cell density at the time of transfection. In general, 30-70% confluency is recommended.
- Transfection method (traditional or reverse transfection).
- Length of exposure of cells to transfection agent-RNA oligonucleotides complexes.
For many miRNA mimics, maximal activity is achieved after 24 hours, but biological assays may be performed 24-72 hours posttransfection. If you maintain your transfected cells longer than 24 hours, we suggest replacing the existing medium with fresh medium at 24 hours posttransfection, for greater viability of the cells.
Using mirVana miRNA Inhibitors to analyze endogenous miRNA function
For the following applications, use a cell line that expresses relatively high levels of the mature miRNA of interest.
General transfection starting points
mirVana miRNA Mimics and Inhibitors typically work best when transfected at a final concentration of 3-30 nM. Optimization experiments might include a concentration range of 1-100 nM. To increase accuracy and reproducibility when preparing transfection complexes, prepare a dilution of your stock oligonucleotides, and pipet a higher volume of diluted stock.
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The turnaround time varies depending on the level of purification and whether it is a custom order or not. Please see the approximate timetable below:
Standard purification (5 and 20 nmol), 4 business days
HPLC/in vivo ready (250 nmol), 15 business days
Custom mirVana Mimics and Inhibitors, 15 business days
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Yes, please provide us with your unpublished miRNA sequence and we can synthesize a corresponding mirVana mimic or inhibitor by clicking on this link (www5.invitrogen.com/custom-genomic-products/tools/mirna/).
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Negative control sequences require a confidential disclosure agreement while target sequences require prior proof of purchase for release.
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Store the dried oligonucleotides in a non-frost-free freezer at or below -20 degrees C. Store resuspended oligonucleotides at or below -20 degrees C for extended periods (up to 1 year), and they can undergo up to 50 freeze/thaw cycles without significant degradation. We recommend aliquoting the resuspended miRNA to prevent contamination. Storage at -80 degrees C is fine but not necessary. Working stock solutions can be stored at 4 degrees C for up to 7 days before use.
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Yes, we offer both positive and negative controls for our mirVana miRNA inhibitors. The mirVana miRNA Inhibitor let-7c Positive Control is intended for use as a positive control for mammalian cell culture experiments using mirVana miRNA Inhibitors (Cat. No. 4464080, 4464081). Endogenous let-7c miRNA has been shown to negatively regulate HMGA2 mRNA in cultured cells. HMGA2 is a ubiquitously expressed, non-histone, chromatin protein that can modulate gene expression through changes in chromatin architecture. let-7c miRNA down-regulates levels of HMGA2 mRNA. When transfected into human and mouse cell lines, let-7c miRNA inhibitor blocks endogenous let-7c miRNA, resulting in increased levels of HMGA2 mRNA. Thus, let-7c miRNA inhibitor activity can be monitored in human or mouse cells using real-time reverse transcription PCR (RT-PCR) to detect HMGA2 mRNA.
mirVana miRNA Inhibitor Negative Control #1 is intended for use as a negative control for experiments using mirVana miRNA Inhibitors (Cat. No. 4464076, 4464077, 4464078, 4464079). Transfect this negative control in parallel with your positive control (such as let-7c Positive Control) and experimental mirVana miRNA Inhibitors. Use target expression from negative control-transfected samples as a baseline for evaluation of the effect of the control and experimental miRNA inhibitor on target gene expression.
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Yes, we offer both positive and negative controls for our mirVana miRNA mimics. The mirVana miRNA Mimic miR-1 Positive Control (Cat. No. 4464062, 4462063, 4462064, 2262065) is designed to mimic mature miR-1 miRNA after delivery to mammalian cells. miR-1 is associated with down-regulation of many genes. In particular, miR-1-mediated down-regulation of Protein Tyrosine Kinase 9 (PTK9) has been shown to occur at the mRNA level. miR-1 Positive Control is intended for use as a positive control for transfection experiments using mirVana miRNA Mimics. Monitor activity of miR-1 Positive Control through its gene silencing of PTK9, using real-time reverse transcription-PCR to detect PTK9 mRNA.
mirVana miRNA Mimic Negative Control #1 (4464058, 4464059, 4464060, 4464061) is designed to use as a negative control for experiments using mirVana miRNA Mimics. Transfect the Negative Control using the same methodology as for your positive control (such as miR-1 Positive Control) and experimental mirVana miRNA Mimics. Use target gene expression from Negative Control-transfected samples as a baseline for evaluation of the effect of the control and experimental miRNA mimic on target gene expression. mirVana miRNA Mimic Negative Control #1 is a random sequence miRNA mimic molecule that has been extensively tested in human cell lines and tissues and validated to not produce identifiable effects on known miRNA function.
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The average molecular weight for mirVana miRNA mimics is 14,000 g/mol (where 1 nmol = 14 µg), and 11,000 - 12,000 g/mol (where 1 nmol = 11 - 12 µg) for mirVana miRNA inhibitors. The exact molecular weights are proprietary.
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Please visit miRBase: The microRNA database (http://www.mirbase.org/).
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We can label our Pre-miR and Anti-miR miRNA, but do not offer labeled mirVana Mimics and inhibitors. 5' modifications include Alexa Fluor 555, Alexa Fluor 647, FAM, and Cy3. Request a quote here (http://www.thermofisher.com/us/en/home/global/forms/custom-sirna-mirna-quote-request.html).
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Yes, submit your unpublished miRNA sequences and/or order custom modified miRNA here (http://www.thermofisher.com/order/custom-genomic-products/tools/mirna/).
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Invitrogen Anti-miR inhibitors & Invitrogen Pre-miR miRNA Precursors are our first generation mimic and inhibitors. Invitrogen Anti-miR miRNA Inhibitors are chemically modified, single stranded nucleic acids designed to specifically bind and inhibit endogenous microRNA (miRNA) molecules. These are ready to use inhibitors that can be introduced into cells using transfection or electroporation. Pre-designed inhibitors have been created for all miRNAs listed in miRBase version 15.0.
Invitrogen Pre-miR miRNA Precursors are small, chemically modified double-stranded RNA molecules designed to mimic endogenous mature miRNAs. These ready-to-use miRNA mimics, which are similar to, but not identical to siRNAs, can be introduced into cells using transfection or electroporation parameters similar to those used for siRNAs. Please note, Pre-miR miRNA Precursors are not hairpin constructs and should not be confused with pre-miRNAs.
mirVana miRNA mimics and inhibitors are our second generation of mimics and inhibitors. mirVana miRNA mimics are small, chemically double-stranded RNAs that mimic endogenous mature miRNAs and enable miRNA functional analysis by up-regulation of miRNA activity. They have enhanced specificity over the first generation mimics, are delivered as HPLC purified, and ready to transfect.
miRNA inhibitors are small, chemically modified single-stranded RNA molecules designed to specifically bind to and inhibit endogenous miRNA molecules and enable miRNA functional analysis by down-regulation of miRNA activity. They have enhanced potency over our first generation inhibitors. Pre-designed inhibitors were created for all miRNAs listed in miRBase version 19.0. They come delivered as HPLC purified, and ready to transfect. Learn more about mirVana miRNA mimics and inhibitors through this video (https://www.youtube.com/watch?feature=player_embedded&v=Q3MO1lmT0kw). Both mimics and inhibitors are available individually or as libraries.
They are compatible with both in vitro and in vivo applications, and have been validated with Lipofectamine RNAiMAX Transfection Reagent for use in cell-based systems, and with Invivofectamine 3.0 Transfection Reagent for in vivo delivery. These oligonucleotides are nontoxic and do not induce an immune response in the animal models tested. In vivo-ready mirVana miRNA mimics and inhibitors have been purified by HPLC and dialysis and are ready for immediate in vivo use. For all library inquiries, please email: rnailibraries@lifetech.com.
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siRNA mediates degradation of a target mRNA molecule in a sequence-specific manner, while miRNA suppresses translation without cleaving mRNA.
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miRNA (microRNAs) are short, highly conserved small noncoding RNA molecules naturally occurring in the genomes of plants and animals. miRNAs are single stranded, 18-25 nucleotides long and regulate posttranscriptional mRNA expression, typically by binding to the 3' untranslated region (3'-UTR) of the complementary mRNA sequence, resulting in translational repression and gene silencing. Studies have shown that thousands of human protein-coding genes are regulated by miRNAs, indicating that miRNAs are “master regulators” of many important biological processes.
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These products are delivered dry lyophilized.
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