pLenti-UTR-Dual-Luciferase Blank Vector
Control lentiviral vector expressing firefly luciferase and renilla luciferase.
|Unit quantity||1.0 μg|
1 year when stored at -20°C or lower in a non-frost free freezer.
- Lentiviral Vector Amplification Protocol
- Selection-Drug Killing Curve
- Lentivirus Packing Protocol (for use with Vectors only)
- Lentivirus Infection Protocol
- UTR Reporters Handbook
- dNTPs (10 mM)
- RNaseOFF Ribonuclease Inhibitor
- 5X RT Buffer
- miRNA Oligo (dT) Adapter
- Nuclease-free Water
- Poly(A) Polymerase, Yeast
- 5X Poly(A) Polymerase Reaction Buffer
- 25mM MnCl2
- ATP (10 mM)
- OneScript® RTase
- Enhanced Lentivirus Safety Features: Replication Incompetency
- Suggested MOI for Common Cancer Cell Lines
|Which species are covered by a miRNA qPCR array?|
The complete layout and miRNA targets included in our arrays can be found on the product page. Currently, ABM offers miRNA qPCR arrays for whole human and mouse genomes, as annotated by miRBase Release 16.0.
|Can I order primer sets for miRNA species not listed on your website?|
Yes, we are able to design and provide primer sets for mature miRNA species not listed on our website. Contact a representative at [email protected] for details. Please provide the appropriate miRNA accession number(s).
|Can I use other cDNA synthesis kits for my sample preparation?|
No, please use ABM's miRNA OneScript cDNA Synthesis kit (Cat No.G269 or G270) to reverse transcribe your RNA. Failure to do so will not yield valid qPCR results. Each primer set includes a universal reverse primer and is specific for the unique and proprietary sequence incorporated into the cDNA by the miRNA adapter in the miRNA cDNA synthesis kit. The miRNA qPCR primer sets cannot accurately detect cDNA generated using other first strand synthesis kits.
|Can precursor miRNAs be detected by the primer sets?|
Each primer set has been designed and tested to detect only mature miRNA. We can alternatively offer primers specific for pre-cursor miRNA as a custom service. The cost of the service is $300.00 for 150ul/10uM. Please contact [email protected] with your specific requirements for a quote.
|I still have additional questions, who do I contact?|
For further details, please email [email protected] or 1-866-757-2414.
|Can ABM provide assistance with summarizing the miRNA qPCR array for my publications?|
Sure, please contact us for publication assistance.
|Where can I view performance data?|
1nt performance data for our high performance arrays can be found on our general qPCR miRNA Array page.
|Does ABM provide a miRNA profiling service?|
Yes, we provide a miRNA profiling service using our latest generation of miRNA arrays. Visit our miRNA custom services FAQs or the miRNA profiling services page for more information.
|What type of RNA internal standards are there for quantitative determination of miRNA?|
Our arrays include 4 endogenous controls for normalization of your qPCR data. The human endogenous controls are snoRD44, snoRD47, snoRD48, and U6-2. For mouse, the endogenous controls are Rt U1, U6, RNU43, and snoRNA142. All our endogenous controls have been validated using multiple sources of RNA.
|Which qPCR machine do I need to read miRNA arrays?|
You may use any 384-well qPCR machine, but remember to specify your machine in your order so your array contains the right master mix for your machine.
|How many miRNA species are included in the arrays?|
Our Human and Mouse Whole Genome miRNA qPCR arrays cover 1034 and 726 miRNA mature species, respectively.
|What reagents do I need to perform a qPCR miRNA array experiment?|
All the reagents you will require for the qPCR miRNA profiling are contained within the array you order from ABM. The kit includes 5ml of complimentary 2X BrightGreen qPCR miRNA mastermix, sufficient for two 384-arrays. Additional mastermix can be ordered online or by contacting [email protected]
|What is the sequence of the universal primer contained in the miRNA qPCR arrays?|
The sequence of the universal primer is proprietary, and unfortunately cannot be disclosed under any circumstances.
|What is in the wells of your qPCR arrays?|
Each well contains 4ul of primer mixture, including miRNA specific and universal primer. Our optimized qPCR Mastermix is provided in a separate tube.
|How many biological replicates do I need for each condition?|
To increase confidence and reduce experimental error, it is suggested that you use at least 3 replicates per sample. Alternatively, you may wish to use only one sample per condition; pick out miRNA of interest using our analysis spreadsheet (or do your own analysis); and then use replicates for select miRNA species. You may contact a representative at [email protected] for quotes and details on individual miRNA assays. Or, order individual miRNA primer sets online.
|Can precursor miRNAs be detected on the qPCR array?|
Each primer set has been designed and tested to detect only mature miRNA.
|Is there a way I can check for the presence of small RNA in my total RNA sample before analyzing?|
Yes, this can easily be done with a 1-2% agarose gel. You should see a 4.5kb band at double the intensity of a 1.9kb band; as well you should not see any smearing, as this is a sign of RNA degradation. If you do not have gel documentation equipment, a similar analysis can be done using a UV spectrometer. Ensure the 260nm/280nm ratio is 1.8, while the 260nm/230nm ratio is >1.0. A 260nm/280nm ratio >2.0 indicates unwanted residual ethanol while a ratio <1.5 suggests RNA degradation or sample contamination.
|Do I need to enrich my total RNA sample for small RNAs?|
No, our miRNA profiling system is designed and optimized to detect all miRNA species starting with total RNA.
|How do I prepare my total RNA sample?|
Trizol reagent is recommended. You may also use other methods as long as it preserves the total RNA including small size RNA within your sample and minimizes the amount of solvent left in the sample.
|In what form are the miRNA primers sets supplied?|
We provide 150ul vials of each primer (1 x forward primer and 1 x universal reverse primer) at a concentration of 10uM.
|I am very interested in your miRNA products. How about the price for the 3' UTR Luciferase/GFP Reporters? Could I directly order with your company or online and pay by PO?|
To search for the price of 3'UTR product, please go to http://www.abmgood.com/microRNA-miRNA/targetvalid.php?csn=26&ssn=11838&dsn=12288, and enter the gene name. You can place your order by email, telephone, fax, mail or online. PO number or Visa can be used. Please see the detail by clicking the following link http://www.abmgood.com/misc/orders.php
|I want some information with regards to your quality methods of the primers to ensure that they are pure.|
Every oligo is deprotected and desalted to remove small molecule impurities. Oligos are quantified twice by UV spectrophotometry to provide an accurate measure of yield.
|What is the shipping temperature for the primers?|
The primers will be shipped with ice packs. If requested, room temperature for 1-2 days is fine but it is not recommended for optimal performance.
|What the Tm for the primer?|
We have validated all the primers with our own miRNA qPCR mastermix. For the qPCR set up instruction, please refer to page 6 of this file http://www.abmgood.com/miRNA/pdfs/miRNA%20HandbookV11.pdf Mastermixes from other companies may be used but the Tm of the primers will slightly vary. The Tm will need to be optimized.
|How does the universal reverse primer work?|
The universal reverse primer can be used for all mature miRNA. To quantify or amplify a specific miRNA, the universal reverse primer with the appropriate forward miRNA primer need to be used. Our forward primers can work with any kits. Our universal primers will only work with our cDNA synthesis kit. http://www.abmgood.com/miRNA-microRNA-cDNA-Synthesis-Kit.html
|How does the universal reverse primer work in amplifying mature miRNA?|
For PCR amplifying mature miRNA, total RNA needs to be isolated and reverse transcribed with our cDNA synthesis kit. A miRNA specific forward primer and an universal reverse primer (that recognizes a specific sequence incorporated to the cDNA) are used to amplify mature miRNA.
|Do I need to purify the sample after adding the poly A tail from the Ambion kit before using the cDNA synthesis kit?|
No, that is not necessary. As per page 4 in the miRNA manual http://www.abmgood.com/miRNA/pdfs/miRNA%20HandbookV11.pdf, the sample can be used directly for the next step in the cDNA synthesis kit.
|How to design individual miRNA primer ?|
The individual miRNA primer could be purchased at http://www.abmgood.com/miRNA-Primer-Sets-Human.html
|How many tests can I perform with the given amount?|
Each primer unit is 10uM and 150ul. Thus, each unit will be approximately 250 qPCR reactions (final concentration of 300nM in 20ul final reaction volume).
|Is it possible to use your kit (miRNA cDNA Synthesis Kit) to do quantitative PCR with the Taqman strategy?|
This kit along with our miRNA primers would not be compatible for use with Taqman strategy.
|What is the source of the Luciferase gene?|
It is from the firefly.
|How does the 3'UTR platform work?|
The stable cell line or the infected cells from the lentivirus will consistently express luciferase/GFP unless there are miRNA that activate the 3'UTR to "knockdown" the reporter expression.
|What is the forward and reverse sequencing primers for the 3'UTR luciferase reporter systems?|
Forward sequencing primer: Luc Forward primer 5'-GCAAGTTGGACGCCCGCAAGATC-3' Reverse sequencing primer: SV40 promoter reverse primer 5`-TAGTCAGCCATGGGGCGGAGA -3'
|If we order primers (e.g. for miR126) Do you provide us with the sequence?|
We do not provide the sequences for our miRNA primers. However we do validate all of our primers in house and can verify their functionality.
|Whats the parental cell line for the 3'UTR stable cells?|
|Are these primers compatible with the precursor form of miRNA?|
Our miRNA primer sets are compatible with the mature form only.
|What is the upper size limit for the 3'UTR insert for viral packaging?|
For GFP, we can fit a 3.5kb insert for efficient packaging. For Luc, we can fit a 2.6kb insert for efficient packaging. The above size limits can be stretched a little (~0.5kb-1kb larger), but the packaging efficiency and virus titer will be lower.
|How does the universal reverse primer work?|
The universal reverse primer can be used for all mature miRNA. To quantify or amplify a specific miRNA, the universal reverse primer with the appropriate forward miRNA primer needs to be used. Our forward primers can work with any kits. Our universal primers will only work with abm's cDNA synthesis kit.
|Can I use SYBR green mastermix for downstream qPCR of the cDNA produced with this kit?|
It may be possible to use a SYBR green mastermix for qPCR, however, our BrightGreen miRNA miRNA qPCR MasterMix recipe has been fully optimized to provide the most successful conditions for qPCR. We have performed extensive testing with minor differences in the composition of miRNA qPCR buffer, this has proven to give very dramatic differences to the results of qPCR. General qPCR mastermixes have more additives to prevent non-specificity and formation of primer dimers and the conditions are often too harsh for the miRNA primers to anneal properly
|Could 3'UTR GFP reporter be used with a conventional transient transfection system (e.s.Lipofectamin 2000)? Could this vector be used to validate the activity of an endogenous overexpressed miRNA?|
Yes, this vector can also be transiently transfected using a transfection reagent such as lipofectamine 2000, or DNAfectin available from abm. This reporter construct can also be used to validate interactions of an endogenously expressed miRNA, if the known target site is present in the 3' UTR sequence.
|Should DNase treatment be performed for the RNA extracted before this procedure?|
You can perform gDNA removal before proceeding with the cDNA synthesis. However, we would recommend using Cat# G488 AccuRT gDNA removal kit. Our product uses proprietary method to removal gDNA and to stop the gDNA removal without using chelating agent such as EDTA and no heating step is involved.
|I have ordered your miRNA primers and am using the Bio-Rad CFX Connect™ Real-Time PCR Detection System. Is your system a 1 color or 2 color system?|
Our EvaGreen miRNA qPCR Mastermixes is EvaGreen based and so the machine can only detect the EvaGreen/SYBR Green Channel. Please note that you will need to use abm's miRNA cDNA synthesis kit (Cat#G269/ G902) in order to use our primers/universal reverse primer.
|Can the miRNA cDNA Synthesis Kits also be used for plant miRNAs?|
Yes, these kits can also be used for plant miRNAs.
|Can this kit be used for circulating miRNA or exosomal miRNA?|
Yes, our kit does work with circulating miRNA and exosomal miRNA. Depending on the abundance of the target gene, you may use a lower template amount. E.g. House-keeping genes starting template of 100ng total RNA may be sufficient.
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