let-7c miRNA Inhibitor
|Description||microRNAs (miRNAs) are small non-coding RNA sequences important for gene regulation. This let-7c miRNA inhibitor is designed to bind to let-7c mature miRNA, inhibiting its effect on the cell. This miRNA inhibitor can be used in experiments to investigate miRNA targets and roles in the cell.|
- Lentiviral Vector Amplification Protocol
- Important Considerations for Lentiviruses
- AAV Packaging Protocol
- AAV Plasmid Amplification Protocol
- Selection-Drug Killing Curve
- Adenovirus Amplification/Transduction Protocol
- Lentivector Packaging Protocol
- Packing Protocol (for use with Vectors only)
- Lentivirus Infection Protocol
- Synthetic miRNA Transfection Protocol
- Enhanced Lentivirus Safety Features: Replication Incompetency
- Suggested MOI for Common Cancer Cell Lines
- miRNA(microRNA) Expression and Detection
- Adeno-Associated Virus (AAV) FAQ
- Adenoviral System FAQ
- Control Vectors and Viruses (Blank, GFP, Scrambled, and more)
|What is the Sanger miRBase Sequence Database?|
miRBase is a sequence database that has been established by the Sanger Institute. Each entry in the microRNA Registry represents a predicted hairpin portion of a microRNA transcript (termed mir in the database), with information on the location and sequence of the mature microRNA sequence (termed miR). The database provides microRNA gene hunters with unique names for novel microRNA genes prior to publication of results and a searchable database of published microRNA.
|When was the latest update of your array sequences?|
The content of our arrays has been updated to miRBase Release 16.0.
|Do you sell any products that allow detection of microRNAs using in-situ methods|
We currently don't have in-situ miRNA detection methods.
|Is there any product literature?|
Cheng, A.M., Byrom, M.W., Shelton, J., et al. (2005). Antisense inhibition of human miRNAs and indications for an involvement of miRNA in cell growth and apoptosis. Nucleic Acids Res. 33, 1290–1297
|How do I detect the reduced level of the miRNA after inhibition?|
Target miRNA is not degraded, but it is silenced by the antisense strand expressed by the inhibitor construct, which will effectively bind and sequester it. Using qPCR to detect changes in downstream target gene expression is therefore recommended. Another method is to use a GFP/luciferase 3' UTR reporter that contains a known miRNA target site to detect changes in reporter expression (http://www.abmgood.com/3%27UTR-Reporter-Cell-Line.html).
|Can RISC-combined-fragment bind/inhibit RISC-combined-(target)miRNA?|
The exact inhibition mechanism is unknown. It is experimentally demonstrated that the expressed anti-sense miRNA will prevent the target miRNA from binding to its gene 3'UTR target.
|How does inhibitory miRNA lentivirus (LentimiRa-Off) work?|
Inhibitory miRNA lentivirus works by expressing anti-sense microRNAs (in siRNA form). There are many references for anti-sense miRNA knockdown properties. The way the inhibitor is processed is more similar to shRNA than miRNA processing, so the flanking native miRNA sequences are not required. The inhibitor (antisense to the mature sequence) will be processed in the shRNA pathway and will knockdown the miRNA as an shRNA would normally knockdown mRNA of its target gene. Please check the following Cheng, A.M., Byrom, M.W., Shelton, J., et al. (2005). Antisense inhibition of human miRNAs and indications for an involvement of miRNA in cell growth and apoptosis. Nucleic Acids Res. 33, 1290–1297.
|what is the difference between miRNA and the miRNA with the asterisk symbol?|
miRNA and miRNA* are the two different double-stranded products from dicer processing of the same precursor miRNA. the miRNA without the asterisk represent the predominant product and the one with the asterisk represent the portion from the opposite arm of the precursor. More information on the names can be found in the link below: http://www.mirbase.org/help/nomenclature.shtml
|Do you have any mutant constructs available for the 3'UTR miRNA reporter vectors for miRNA-mRNA interaction validation?|
We do not have any mutant constructs in our inventory, all the listed constructs are wild type. We can offer mutant constructs as a custom service. The exact sequence to be mutated must be supplied by the end-user in each case. Please contact [email protected] with any specific requests.
|Do I need to use an internal control primer for quantitative detection of miRNAs (the same as housekeeping primers which we use in qPCR for mRNAs detection)?|
For human miRNA plates, the controls are as follows: 1) SNORD44, cat MPH00003 2) SNORD47, cat MPH00004 3) SNORD48, cat MPH00005 4) U6-2, cat MPH00001
|After cell transduction with the vector,should I dedetect pri-miRs or mature miRs?|
The mature miRs could be detected after transduction/infection.
|What promoter is the miRNA under?|
The miRNA is being expressed through a CMV promoter.
|What is the genetic structure of the miRNA off insert?|
The off-insert is expressed by the H1 promoter and will form a stem loop structure like immature shRNA that is complementary to the targeted miRNA. We take the antisense (the inhibitor) to the mature miRNA sequence and form a hairpin structure with the mature miRNA sequence (has several mutations in it to render it inactive) after transcription. The inhibitor gets processed in the same pathway as regular miRNA precursors and the resulting antisense inhibitor actively knocks down mature miRNA. The structure of the inhibitory insert goes like this: mature miRNA sequence (with mutations) - Hairpin loop - Antisense miRNA sequence (inhibitor) - TTTTTT termination sequence.
|What sequencing primers can I use?|
The reverse sequencing primer is 5'-GGAACATACGTCATTATTGACGTC-3'. A forward primer is 5'-GTGTCACTAGGCGGGAACAC-3'.
|How does miRNA inhibitor adenovirus work?|
The miRNA inhibitor is in the form of a short hairpin which is complementary to the mature miRNA sequence that it targets. Its expression is under the control of the H1 promoter in an Adenovirus vector. The packaged adenovirus can be used to transfect your target cells and deliver the miRNA inhibitor into the cells and knockdown its target mature miRNA.
|Can I quantify mature miRNA in total RNA using your cDNA synthesis and kit and qPCR master mix?|
Yes, that is what it is designed for.
|In the manual, the vector map is shown with mature miRNA flanked by miR-30 (mir BB part of the vector map) to form the premature miRNA. What happens to the miR-30?|
There will be no final mir-30 expression. The miR-30 (also known as mir BB in the map) remains in the vector backbone and is used for miRNA processing. The final miRNA expressed is the mature target miRNA only. Reference: Cullen et. al. 2003. Sequence requirements for micro RNA processing and function in human cells. RNA. 9(1): 112–123. Guo et al. 2009. Construction of an Artificial MicroRNA Expression Vector for Simultaneous Inhibition of Multiple Genes in Mammalian Cells. IJMS.
|Which genes are targeted by a specific miRNA?|
You can search for predicted and validated miRNA target genes at http://mirbase.org/. Just type in your miRNA name (eg. hsa-mir-145) or accession number in the search bar and look for the targeted genes.
|Are these vectors supplied lyophilised or in solution?|
Our miRNA lentivectors are provided as 500ng DNA in 10ul storage buffer solution composed of 10mM Tris-HCI, 1mM EDTA, pH 8.0.
|What promoter drives the inhibitory miRNA?|
|Does the profiling (qPCR) require a specific instrument or it can be done by every real time PCR instrument?|
We have a range of miRNA qPCR mastermixes available in optimized formulations for all qPCR machines, please see our web page http://www.abmgood.com/miRNA-qPCR-Mastermixes-microRNA-qPCR.html
|is there a reporter gene for the miRNA adenovirus?|
All of the miRNA adenoviruses have a GFP reporter gene.
|Is there a reporter gene for the miRNA inhibitory adenovirus?|
All of the miRNA inhibitory adenoviruses have a GFP reporter gene.
|What insertion/restriction sites are used to clone the inhibitory miRNA sequence into the vector?|
The sites used for all miRNA inhibitor constructs are XbaI/XhoI
|pLenti-III-miR-Off system or LentimiRa system uses blank vector for control, but I usually use scrambled control for si-RNA experiments. Is it supported to use blank control not scrambled for miRNA experiments?|
Both scrambled and blank controls are effective negative controls. However, there are debates over off-target effects when using scrambled sequences. By using a blank vector as negative control, the off-target effect can be eliminated. If your experiment requires a scrambled control over a blank control, we can custom make that for you as well upon request.
|Why are miRNA mimics on your site offered at 2 x 2.5nmol, but miRNA inhibitors at 2 x 5.0nmol?|
This is because our mimics now are double-stranded while the inhibitors are single-stranded. The amount of materials are quantified in OD and both mimics/inhibitors are synthesized to yield the same OD. Therefore, the number of moles of mimics is half of that of the inhibitors.
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