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.
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, starting from $375 per mutant (depending on the length of the insert and the complexity of the mutation(s) required).
The exact sequence to be mutated must be supplied by the end-user in each case.
Please contact email@example.com 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
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.
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.
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
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.
How can we identify which of the genes are affected by miRNA?
There are targets confirmed by experimentation, and predicted targets that are unconfirmed. Predicted targets are just that - prediction by software analysis of sequence, composition, thermodynamic factors, 3D conformation, etc.
We can provide additional bioinformatics service where we generate a list of gene targets. It is important to note that the gene targets need to be systematically tested.
The 3'UTR gene reporters transduced with miRNA expression vectors will be the test to confirm biological activity of predicted targets. If the miRNA knocks down that expression, it is generally considered that the miRNA targets the gene where the UTR reporter was derived from.
Rescue with the knockdown (our 'OFF' products) or a synthetic target is generally the confirmatory step.
What is the recommended way to extract RNA?
We recommend that you use TRIzol as your RNA isolation method to ensure high quality and to retain all miRNA species in each sample. The RNA can be sent frozen in DEPC-treated /RNase-free ddH2O with a minimum concentration of 350ng/ul and packed securely in dry ice. Alternatively, you may send us your RNA pellet stored in 75% ethanol and packed in dry ice, but please clearly indicate that your samples are stored in ethanol. Please ship your samples using overnight delivery service and do not ship on Friday, or your samples will thaw before we are able to receive them.
Can I use other cDNA kits to prepare the samples for you?
Our miRNA array requires a special adaptor on the cDNA, which can only be added with our cDNA synthesis kits (cat#G269 and cat#G270).
Do you have references on your technology?
Our miRNA array was developed in June 2010 and most articles take about 2-3 years for publication. Thus, the articles are currently in progress. However, we do have a list of satisfied customers who can confirm the success of the service with their samples. We will be happy to provide the list upon your request. Finally, we can work on two samples and then send the data to you. If you are pleased with the results, we can then work out for the remaining samples.
What is my Login ID?
Your Login ID is the same as your Customer ID number and can be found on your invoice/billing form.
What miRNA species are covered by the profiling service?
Our profiling service includes 1034 and 726 mature miRNA for human and mouse, respectively.
When I log in to the database, some of the links go no where.
Either your results are not ready or the type of service you have ordered does not include these types of files. We will send you an email once your results are ready for viewing and download.
Full profiling services usually include two excel spreadsheets but no service report. Custom assays include a service report but no excel sheets.
What is my Access Code?
Your access code will be emailed to you once your results are available. If you have lost your access code, please contact a representative.
Are individual custom assays available?
Yes, we can perform custom services for specific miRNA species. Contact a ABM representative at order@abmGood.com for quotes and details.
Can I use other cDNA synthesis kits for my sample preparation?
If you are sending cDNA, please use ABM miRNA EasyScript cDNA Synthesis kit (Cat No.G269 or G270) to reverse transcribe your RNA. Failure to do so will not yield valid qPCR results. The universal primer in every assay of the miRNA qPCR profiling service 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 arrays cannot detect cDNA generated using other sources of first strand synthesis kits.
I still have additional questions, who do I contact?
For further details, please email firstname.lastname@example.org or 1-866-757-2414.
How long will it take?
The full profiling service, starting from cellular samples, can be completed within 12 business days. For orders containing many multiple samples, please contact a representative.
What about unused sample?
After completion of the experiment you may either ask ABM to safely dispose of your sample, or if you wish to receive unused portions of valuable samples back, this can be arranged at your own expense.
What does ABM do with the data?
We will keep the data secure on our servers for a year, allowing you repeated access in case of data loss on your end. However, after a year all data will be deleted and scrambled rendering it irrecoverable, so please retrieve and save your data prior to this date.
Can I analyze my data myself?
Yes, contained within the data spreadsheet we provide is both raw and normalized data; you can extract these and proceed with your analysis however you prefer.
How does ABM conduct the experiment?
Samples will first have their RNA isolated using our in-house standard procedures. Then we will synthesize cDNA from the RNA isolate and this will be used for the qPCR plate miRNA analysis.
What data will I receive from ABM?
Your results will contain:
1. Raw Ct values for 1034 or 726 miRNA for human or mouse, respectively
2. Raw Ct values for 4 endogenous controls for normalization
3. Normalized and processed values to help distinguish differences in microRNA levels between your sample and control
4. A scatter plot of points derived from fold changes between your control and sample
5. An excel data analysis program for easy sorting and analysis of your samples
**You can choose to view your data in its analyzed form or pull the data and analyze it yourself.** .
How do I receive the data?
Data can be downloaded from a secure login site on our website using a login and password provided with each order. Alternatively, a file of the data can be emailed upon request.
Where do I send my samples?
Applied Biological Materials
#1-3671 Viking Way
Send all samples
1. Using overnight delivery
2. Never on a Friday
3. Frozen and packed securely in dry ice.
**Check that all form necessary to clear customs are sufficiently filled out and included**
How much sample is needed?
There are three options for sending samples to us:
1. Send us cell pellets containing a minimum of 10^7 cells. As a general rule, cells from a minimal of 10cm dish at over 80% density or 10-20mg tissue should be enough.
2. Send us at least 0.5ug of total RNA or 100ng small RNA - we suggest using TRIzol for isolation. Minimum concentration of 100ng/ul .
3. Send us cDNA, synthesized from 0.5ug of RNA using our miRNA cDNA synthesis kit (cat. no. G269 or G270).
How do I prepare my RNA samples for submission?
1.Please send us a minimum of 0.5ug total RNA (recommended). Alternatively, you may send us 100ng of small RNA.
2.We recommend that you use TRIzol as your RNA isolation method to ensure high quality and to retain all miRNA species in each sample.
3. Please send your RNA frozen in DEPC-treated /RNase-free ddH2O with a minimum concentration of 100ng/ul and packed securely in dry ice . Alternatively, you may send us your RNA pellet stored in 75% ethanol and packed in dry ice, but please clearly indicate that your samples are stored in ethanol.
4. Ship your samples using overnight delivery services and do not ship on Friday, or your samples will thaw before we are able to receive them.
**If you are shipping from outside of Canada please ensure that all appropriate forms necessary to clear customs are included or your sample will be delayed at the border. You may wish to send your total RNA stored in ethanol instead of ddH2O to better maintain sample integrity.**
How do I get started?
First determine how many samples you are using in your miRNA profiling project. Then decide whether you want to send us your samples in cell pellet or isolated total RNA form. Isolated total RNA is preferable over cell pellets, and an extra charge for RNA isolation will apply if you decide to send cell pellets or tissues.
Alternatively, you may purchase our miRNA cDNA Synthesis kit (Cat. No. G269 or G270) and synthesize the cDNA template yourself.
For further information, please feel free to email us at technical@abmGood.com or call us at 1-866-757-2414 for further assistance.
Is it necessary to perform poly-A tailing before I send RNA samples?
No, our miRNA cDNA synthesis service includes poly-A tailing. No additional fees for poly-A tailing will apply if you are ordering cDNA synthesis service. However, if you wish to perform poly-A tailing yourself, please clearly indicate that you have done so on your samples.
How many technical replicates does the service include?
Each profiling service includes 1 run across all miRNA profiling plates. For the full list of miRNA species covered, please refer to our product specification sheet:
Duplicates of all house-keeping genes (i.e. SNORD44, SNORD47, SNORD48 and U6-2 for Human profiling service) are run for each plate to ensure accurate and proper loading procedure.
We recommend that you use our profiling service to determine your miRNA of interest. Once you have determined which miRNA species you are interested in, you may order our "Assay of Single miRNA of your Choice" custom service (C205) for further technical replicates or order our miRNA primer sets to perform the experiment yourself.
Can the control miRNA primers from ABM be used for both human and mouse samples?
No, the internal controls (housekeeping primers) are different for human and mouse samples. ABM offers a complete set of internal controls for both species.
For human, the controls are as follows:
1) SNORD44, ABM cat# MPH00003
2) SNORD47, ABM cat# MPH00004
3) SNORD48, ABM cat# MPH00005
4) U6-2, ABM cat# MPH00001
For mouse, the controls are as follows:
1) Hm/Ms/Rt U1, ABM cat# MPM00006
2) U6 snRNA, ABM cat# MPM00002
3) RNU43 snoRNA, ABM cat# MPM00003
4) snoRNA142, ABM cat# MPM00004
For more information, please visit http://www.abmgood.com/miRNA-qPCR-Profiling-Kits.html.
What is the detection limit of your profiling service?
10-100 copies of miRNA present in the sample
Do I need to enrich my total RNA sample for small RNAs?
No, our probes are designed to detect all miRNA species equally; therefore enriching your sample in small RNAs may result in biased data output.
Can precursor miRNAs be detected on the qPCR array?
Each primer set has been designed and tested to be able only to detect mature miRNA.
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.
What is the sequence of the universal primer contained in the miRNA qPCR arrays?
We cannot disclose the sequence of our Universal Reverse Primer, as this is a proprietary design.
Which species are covered by a miRNA qPCR array?
Please refer to the product page for specific arrays we currently offer ( http://www.abmgood.com/miRNA/mirnaarray.php?csn=26&ssn=11839&dsn=12259).