miRNA FAQs
1. Can I create multiple gene carts? Can I email them to someone?
That functionality is not available at this time, but we are hoping to expand the utility of the Gene
Cart with future releases.
2. Can Customer Service representatives see my Gene Cart in the same way they can see my
shopping cart?
The Gene Cart contents do not appear in our internal systems in the same way as the shopping cart.
A Gene Cart is, however, perpetuated in the same way as your shopping cart. If you are logged in
with a valid Dharmacon account, or continue an anonymous session without deleting cookies, the
Gene Cart will be available during subsequent sessions.
3. Why do I need to use a Non-targeting control shRNA with my SMARTchoice Inducible
shMIMIC microRNA?
It's a good idea to use a non-targeting control even if there is little of no shMIMIC microRNA
expression in the absence of doxycyxline. Although doxycycline is generally accepted as “inert,”
some cellular phenotypes, such as reduced growth rate, may be observed upon exposure to
doxycycline. In addition, fluorescent reporters such as TurboGFP and TurboRFP can cause some
subtle changes in cellular phenotypes. To rigorously control for both of these effects, it is
necessary to use a vector-matched Non-Targeting Control shRNA to identify true mature
microRNA-dependent phenotypes.
4. Can I use tetracycline to induce expression of the mature microRNA in SMARTchoice
Inducible shMIMIC microRNAs?
No, Tet-On 3G has been optimized for binding doxycycline, and it responds poorly to tetracycline.
5. Can I export my gene cart?
Export functionality is not available from the Gene Cart. Our instance of Ingenuity is for the
support of gene-based products, not as a standalone bioinformatics tool. A full suite of tools to
utilize the Ingenuity annotations and functionality are available via licensing of the Ingenuity
Pathway Analysis tool (IPA) at www.ingenuity.com.
6. Why are there so many items with “No results” when I send my cart contents to the
Cherry-pick Plater?
The Gene Cart will accept items from Ingenuity that may be part of a pathway or interaction that
are not valid when mapping them to specific Human or Mouse gene-based siRNA and microRNA
products (genes assigned to other species, drugs, gene families, etc.). You can control the types of
molecules added to the Gene Cart by using the Filter or Highlight checkboxes within the Ingenuity
views (Interaction Net and Biological Pathways
7. What is the best method to transfect miRIDIAN microRNA Mimics and Inhibitors?
The miRIDIAN microRNA Mimics and Inhibitors can be delivered using standard lipid-mediated
or electroporation delivery methods. The optimal methods and conditions for delivery will depend
on the specific cell line or cell type that you are using. They should be similar to those for the
delivery of other small nucleic acids (e.g. single-stranded oligos or siRNAs) into the same cell line.
DharmaFECT Transfection Reagents can be used to transfect mimics and inhibitors. Cell
line-specific transfection conditions are available in the DharmaFECT General Transfection
protocol.
8. How can I find the HA-tagged insertion site?
The insertion site can be found in the details section on our internal query. The number given is
the amino acid location where the tag was inserted.
9.What is the yeast Y800 genotype (HA-tagged background)?
The yeast Y800 genotype is as follows: [MATa leu2-D98cry1R/MATalpha leu2-D98CRY1
ade2-101 HIS3/ade2-101 his3-D200 ura3-52 caniR/ura3-52CAN1 lys2-801/lys2-801
CYH2/cyh2R trp1-1/TRP1 Cir0] carrying pGAL-cre (amp,ori, CEN, LEU2) Burns, N., Grimwade,
B., Ross-Macdonald, P.B., Choi, E.-Y., Finberg, K., Roeder, G.S., and Snyder, M. 1994.
Large-scale characterization of gene expression, protein localization and gene disruption in
Saccharomyces cerevisiae. Genes & Dev. 8:1087-1105.
10. What is a the star-strand?
Some precursor microRNAs give rise to two functional mature strands, the primary mature strand
and the minor *-strand (star-strand). These represent the two arms of the stem in the precursor
hairpin and are typically designated by the absence or presence of a ""*"" or by -5p and -3p after
the miR designation for the mature sequence (e.g. hsa-miR-214 and hsa-miR-214* or
mmu-miR-290-5p and mmu-miR-290-3p). These can be easily distinguished by the mature
sequence. Design features of both the miRIDIAN Mimics and Hairpin Inhibitors allow you to
study the function of each mature miRNA separately. The double-stranded Mimics have been
modified to preferentially load the mature strand into RISC, while the Hairpin Inhibitors are
designed to potently bind and inhibit the mature-strand of the endogenous miRNA. For more
detail on miRNA nomenclature, please see www.mirbase.org.
11. How should I store plasmid DNA for my shRNA construct?
We store plasmid DNA for shRNA at -20C. We have had no problems with DNA degradation from
multiple freeze/thaw cycles.
12. What licenses are required for commercial customers to work with the TRIPZ and
pLemiR vectors?
The TRIPZ vector utilizes a Tet-On system which is licensed from Tet Systems Holding GmbH &
Co. KG. Commercial customers cannot purchase the product without a license from Tet Systems
Holding GmbH & Co. KG. The TRIPZ and pLemiR vectors utilizes a TurboRFP marker which is
licensed from Evrogen. Commercial customers can purchase the TRIPZ/pLemiR products and use
them for 6 months without a license from Evrogen. After that time the commercial customer will
require a license from Evrogen or be required to cease to use the product.
13. How long does inhibition last after the transfection of miRIDIAN microRNA Inhibitors?
The duration of inhibition depends greatly on how well-expressed a mature microRNA is in a
particular cell line. Inhibitors generally last longer than 48 hours in cells. The inhibitors are
stabilized by their chemical modification pattern and have performed well in-house to at least 96
hours after transfection. We would suggest a time-course experiment to determine, empirically,
how long they will remain potent in your system.
14. What is a SIN vector?
A Self-Inactivating (SIN) vector is a retroviral vector that contains a non-functional or modified 3'
Long Terminal Repeat (LTR) sequence. This sequence is copied to the 5' end of the vector genome
during integration, resulting in the inactivation of promoter activity by both LTRs.
15. Can I build a Gene Cart and order the Cherry-pick Library at a later time?
A Gene Cart is perpetuated in the same way as your shopping cart. If you are logged in with a
valid Dharmacon account, or continue an anonymous session without deleting cookies, the Gene
Cart will be available during subsequent sessions.
16. Can I order shRNA or ORFs based on my Gene Cart entries?
At this time the Gene Cart only supports automatic ordering of siRNA and microRNA reagents via
the Cherry-pick Library Plater. Please contact Technical Support for assistance on ordering
vector-based products based on a gene list.
17. Should I use polypropylene or polystyrene plates to store my library (siRNA, miRNA,
shRNA, cDNA, ORF)?
There is a trade-off between polypropylene and polystyrene for storage at -80C. With
polypropylene, the plates can warp making use with automated/robotics systems problematic.
With polystyrene, the plate can be very brittle while frozen at such a low temperature, but the
problem is not permanent - once the plate is thawed it is not as brittle any longer. So you must
decide which features are more important to you. We tend to use polystyrene for most things as it
is more conducive to our automated manufacturing environment. For synthetic siRNA and miRNA
libraries, a storage temperature of -20C can be used to minimize the problems associated with
storage of plates at lower temperatures.
18. Where can I learn more about miRNA?
You will find a list of frequently cited miRNA-related articles from peer-reviewed journals and
scientific
texts
here:
http://www.thermoscientificbio.com/uploadedFiles/Resources/mirna-reading-list.pdf You can find
a list of references that have used our miRIDIAN miRNA Mimic and Hairpin Inhibitors in vitro
here:
http://www.thermoscientificbio.com/uploadedFiles/Resources/miridian-mimics-inhibitors-negativ
e-controls-product-insert.pdf You can also find a tech note on using Mimics and Inhibitors for
microRNA
functional
analysis
here:
http://www.thermoscientificbio.com/uploadedFiles/Resources/mimics-inhibitors-tech-note.pdf
19. Can I order a custom miRNA miRIDIAN mimic or inhibitor?
Yes, we do offer custom synthesis services for miRIDIAN products. We can work with you to
design miRIDIAN products to complement miRNAs not currently represented as catalog items.
We can also customize our current catalog items with a variety of modifications to meet your
research needs. Please contact our Technical Support department at ts.dharmacon@ge.com for a
customized price quote.
20. Where can I find general literature citations for RNA interference?
Follow
the
link
for
Recommended
Reading
in
the
Support
center:
http://thermoscientificbio.com/resource-library/?type=17179869724&subtype=12884902121
.
Several reading lists covering important topics in siRNA research as well as miRNA research are
available.
21. Can I deliver miRIDIAN microRNA Inhibitors with penetratin (peptide mediated
delivery)?
Although we have not tested penetratin-conjugated inhibitor delivery, we have no indication that
such a conjugation would not work.
22. How long does inhibition last after the transfection of miRIDIAN microRNA Inhibitors?
The duration of inhibition depends greatly on how well-expressed a mature microRNA is in a
particular cell line. Inhibitors generally last longer than 48 hours in cells. The inhibitors are
stabilized by their chemical modification pattern and have performed well in-house to at least 96
hours after transfection. We would suggest a time-course experiment to determine, empirically,
how long they will remain potent in your system.
23. What is the average molecular weight of a miRIDIAN microRNA Hairpin Inhibitor?
18,500 g/mol
24. What is the best method to transfect miRIDIAN microRNA Mimics and Inhibitors?
The miRIDIAN microRNA Mimics and Inhibitors can be delivered using standard lipid-mediated
or electroporation delivery methods. The optimal methods and conditions for delivery will depend
on the specific cell line or cell type that you are using. They should be similar to those for the
delivery of other small nucleic acids (e.g. single-stranded oligos or siRNAs) into the same cell line.
DharmaFECT Transfection Reagents can be used to transfect mimics and inhibitors. Cell
line-specific transfection conditions are available in the DharmaFECT General Transfection
protocol (see Table 2). Concentration and time curves should always be conducted. Suggested
concentrations include 100 nM, 50 nM,10 nM, 2 nM, 0.4 nM, while suggested time points are 24,
48 and 72 hours. However, ideal concentrations must be empirically determined for each
microRNA mimic or inhibitor tested in each cell type, as for siRNA experimentation.
25. What kind of buffer should be used to rehydrate miRIDIAN microRNA Mimics or
Inhibitors?
The miRIDIAN microRNA Mimics, Inhibitors and Negative Controls should be used under
conditions that are compatible with RNA-based oligonucleotides. Thus, resuspending and storing
them in 1X siRNA buffer (diluted from 5X siRNA buffer) is appropriate. Other RNAse-free
solutions that are buffered at a neutral pH may also be used as well.
26. What is the average molecular weight of a miRIDIAN microRNA Hairpin Inhibitor?
18,500 g/mol
27. What kind of buffer should be used to rehydrate miRIDIAN microRNA Mimics or
Inhibitors?
The miRIDIAN microRNA Mimics, Inhibitors and Negative Controls should be used under
conditions that are compatible with RNA-based oligonucleotides. Thus, resuspending and storing
them in 1X siRNA buffer (diluted from 5X siRNA buffer) is appropriate. Other RNAse-free
solutions that are buffered at a neutral pH may also be used as well.