Questions about our products

  • How can I best order custom mRNA at RiboPro?

    We offer several ways to order custom mRNA at RiboPro and you may choose the one that works best for you:

    1. You may order directly via our website. The RNAssembler™ guides you through all the steps and options to design your own mRNA. Not sure about the end-result? Request one of our RiboProfessionals™ to have a look and if needed discuss with you further.
    2. You may contact our RiboProfessionals™ to help you design your mRNA from scratch, taking into account all relevant parameters of your experiment or application.

    In either case, after you order, you will receive an order confirmation stating all the details you may need. Based on the official quotation that you may generate via the website or via your RiboProfessional™, you may arrange a Purchase order at your institution or company to arrange payment. Alternatively, you may pay via the website directly, or discuss with your RiboProfessional™ alternative payment options.

  • How does your cap compare to other suppliers?

    The vast majority of suppliers of custom mRNA use the same basic methodologies as we do; co-transcriptional capping with ARCA, or post-transcriptional enzymatic capping. As such, they are bound to the limitations described for each method. RiboPro’s unique ARCA-Amplify™ protocol increases ARCA capping efficiency by up to 20%, which can have boost translation much more than 20% because of a lower intracellular innate immune response.

    Interestingly, even when similar capping efficiency is achieved, ARCA-Amplify™ outperforms vaccinia capping enzyme attached caps by as much as a factor of 2 in terms of protein expression.

    ARCA-Amplify™ also performs well compared to di-nucleotide cap analogues, which were designed to improve the capping efficiency versus yield trade-off during co-transcriptional capping. The benefit of ARCA-Amplify™ is that it is less restricted in the starting nucleotide than the dinucleotide versions, and can achieve equally high capping efficiencies.

  • How is capping efficiency determined?

    Capping efficiency can be determined with various methods, often requiring digestion of the mRNA, including HPLC, LC-MS, radio-active or fluorescent labelling of the cap. None of these methods are quick or easy to perform, and be thus be costly.

    Therefore, RiboPro has developed a proprietary method that quantifies the capping efficiency. At the moment, we are performing final validation of this method, before it will be available as add-on service for each custom mRNA you order.

    Question is whether capping efficiency is a relevant parameter. When using Vaccinia capping enzyme, it for sure is, as secondary structure at the 5’UTR can prevent or slow the enzyme to add the cap, leaving a potentially significant portion of the mRNA molecules uncapped. When using co-transcriptional capping, the capping efficiency can be controlled by the ratio to the corresponding canonical nucleotide (usually G). In the latter case, capping efficiency is not a function of the 5’ UTR (structure) and as such capping efficiency is predictable. When using co-transcriptional capping, we do not recommend to test for capping efficiency.

  • How is the A-tail added and how do you determine A-tail length?

    Currently, we offer 3 methods of adding the A-tail to the RNA:

    1. Enzymatic addition. The length of the A-tail is controlled via the reaction time, enzyme concentration and substrate concentration. This method produces a Gaussian distribution of A-tail lengths around the desired length, typically with a standard deviation of ~10nt.
    2. Addition by T-tail reverse primer during the PCR reaction as part of the DNA prep. The DNA template contains a predefined number of T-nucleotides that allow for A-tail addition during regular transcription with bacteriophage RNA polymerase. This method produces a highly defined A-tail length.
    3. Addition by inclusion in a cloned construct already containing a defined A-tail. This method produces a highly defined A-tail length.

    The advantage of the PCR based A-tail addition to the DNA template is the uniformity of the A-tails and a cost benefit during synthesis. However, the price for long primers is significant and optimization of the PCR may be required. Therefore, RiboPro only uses this method on catalogue 3’UTRs, unless specifically requested. Additional costs will incur.

    Regardless of the method of A-tail addition, we determine the percentage of transcripts modified and the estimated length of A-tail by a denaturing agarose gel.

  • How is the mRNA capped at RiboPro?

    The cap is often an essential structure for mRNA function; unless an IRES or similar sequence is present, the cap is needed to engage the ribosome and initiate protein synthesis. With other words, without a cap, no protein. RiboPro uses 2 methods of cap addition to the mRNA:

    1)  Co-transcriptional capping; where an anti-reverse cap analogue (ARCA or similar) is added during the synthesis. This method is particularly suited for RNA sequences with a highly structured 5’UTR, since the cap is added before the structured part is synthesized. This method does impact yield and often results in around 80% of transcripts being capped. Uniquely to RiboPro, we have developed a method to increase the capping efficiency up to >99% using ARCA without adversely affecting yield. We call this method ARCA-Amplify™. Interestingly, ARCA-Amplify™ results in higher expression compared to ARCA-cap by other manufacturers and

    2) Post-transcription enzymatic capping; where the purified RNA is heat denatured and the cap is subsequently added by a cocktail of enzymes and substrates. This process is more expensive, but does not affect the yield and often results in >95% of transcript being capped.

    It is worth to note that uncapped transcripts possess a triphosphate at the 5’end, which can be a substrate for the cytosolic RIG-I sensor. Therefore, a high capping efficiency ensures not just many translation-capable mRNAs, but also prevents RIG-I mediated innate immunity, which can negatively affect cellular health and protein synthesis.

  • I ran my RNA on agarose gel and it looks smeared. Why?

    RiboPro performs all necessary QC to guarantee identity and an intact product, therefore it is unnecessary to perform such a gel yourself from a QC point of view. Nonetheless, if you run a gel for another reason, you may encounter smearing.

    Smearing of RNA on an agarose gel may have several reasons.

    1. Not using a denaturing gel. RNA fold back on itself by default and can form complicated secondary structures. If multiple secondary structures are present (as is often the case) they run differently on an agarose gel, unless a denaturing agent is used. Use bleach (1% vol/vol), formaldehyde or another denaturing agent in gel and during sample preparation. RNA denaturing requires heating for a short time in a metal/ion-free buffer containing the denaturing agent.
    2. RNAse activity. All RiboPro products are certified RNAse-free. However, from the moment of opening the tube, RNAses may enter the product and may start destruction of the RNA. This also happens, albeit slower at low temperatures and even in a frozen state. An additional source of RNAse is the agarose gel itself.
    3. Heating the RNA above 55ºC with (trace) metals and ions present in the solution. In the presence of common metals and ions like magnesium, RNA is very unstable at elevated temperatures. If your procedure requires heating the RNA, do so in the presence of EDTA and under buffered conditions.
    4. When enzymatic A-tailing is used, the RNA frequently smears slightly due to the variation in A-tail length that is added to each individual transcript by the enzyme.
  • Is OTS-RNA free of dsRNA contaminants?

    Unless otherwise stated, all our off-the-shelf RNAs in the RNArchive™ are free from detectable dsRNA. Given the enormous inhibition on protein translation (both of specific mRNA and the global mRNA pool in the cell) and the pro-inflammatory cytokine release, we have chosen to taken measures against dsRNA contamination by default.

  • Is your RNA free of dsRNA?

    Unless otherwise stated, all our RNAs in the RNArchive™ are free from detectable dsRNA. Given the enormous inhibition on protein translation (both of specific mRNA and the global mRNA pool in the cell) and the pro-inflammatory cytokine release, we have chosen to taken measures against dsRNA contamination by default.

    For custom RNA synthesis, it is up to you to select dsRNA reduction as an option during synthesis. For the performance of your RNA, we highly recommend this, although this involves some additional costs.

  • What A-tail length do you recommend?

    The optimal A-tail length differs from application to application, however, for the majority of situations, the most optimal A-tail length is between 120-150nt. Contact your RiboProfessional™ to discuss what may work best for your application.

    If you have a particular RNA design in mind that requires a specific A-tail length; RiboPro offers any A-tail length between 10 and 500nt.

  • What is dsRNA and where does it come from?

    During in vitro transcription, bacteriophage RNA polymerases, such as T3, SP6 and T7 RNA polymerase, are frequently used. Although simple in production and use, these polymerases are well-known to produce side-products. These include short RNAs of only a few nucleotides length, produced during abortive cycling, as well as dsRNA. dsRNA production can be a consequence of short RNA fragments acting as primer for RNA-template RNA synthesis, or from self-annealing of the 3′ end of the nascent RNA strand.

    Several counter measures have been developed over time, including HPLC-purification, cellulose-based purification, optimized reaction conditions and modified RNA polymerases. RiboPro uses a proprietary process that prevents dsRNA from contaminating your desired ssRNA product. Our method is on par, or better than the methods used by the competition.

  • What is the standard A-tail length for off-the-shelf products?

    For the majority of situations, the most optimal A-tail length is between 120-150nt. RiboPro uses on all standard off-the-shelf (m)RNA products, 150nt A-tail. Longer A-tails may be requested, at additional cost.

  • What shipping options do you offer?

    Currently, the consensus is that shipping in frozen state on dry ice is the safest way to transport the fragile RNA. Therefore, we typically send the RNA in syrofoam boxes with 10-15kg of dry ice (depending on destination) with FedEx. The costs for shipment are additional to the price listed because of large variations depending on the destination. We always strive to limit the cost for shipping. Depending on the destination, we only ship dry ice on Monday or Tuesday to ensure arrival before the weekend and in a frozen state.

    To mitigate these costs and reduce turn-around time, RiboPro is currently investigating several alternative shipping solutions; allowing the RNA to be shipped at -20oC., 4oC., or even room temperature. Of course, RNA performance is a key outcome in these tests.

    As a customer, you will always have the choice between aforementioned alternative shipping options and dry ice shipping.

Questions about the process

  • How can I best order custom mRNA at RiboPro?

    We offer several ways to order custom mRNA at RiboPro and you may choose the one that works best for you:

    1. You may order directly via our website. The RNAssembler™ guides you through all the steps and options to design your own mRNA. Not sure about the end-result? Request one of our RiboProfessionals™ to have a look and if needed discuss with you further.
    2. You may contact our RiboProfessionals™ to help you design your mRNA from scratch, taking into account all relevant parameters of your experiment or application.

    In either case, after you order, you will receive an order confirmation stating all the details you may need. Based on the official quotation that you may generate via the website or via your RiboProfessional™, you may arrange a Purchase order at your institution or company to arrange payment. Alternatively, you may pay via the website directly, or discuss with your RiboProfessional™ alternative payment options.

  • What is dsRNA and where does it come from?

    During in vitro transcription, bacteriophage RNA polymerases, such as T3, SP6 and T7 RNA polymerase, are frequently used. Although simple in production and use, these polymerases are well-known to produce side-products. These include short RNAs of only a few nucleotides length, produced during abortive cycling, as well as dsRNA. dsRNA production can be a consequence of short RNA fragments acting as primer for RNA-template RNA synthesis, or from self-annealing of the 3′ end of the nascent RNA strand.

    Several counter measures have been developed over time, including HPLC-purification, cellulose-based purification, optimized reaction conditions and modified RNA polymerases. RiboPro uses a proprietary process that prevents dsRNA from contaminating your desired ssRNA product. Our method is on par, or better than the methods used by the competition.

  • What is your turn-around time?

    For custom RNA synthesis our turn around time is between 2-4 weeks. Rarely, specific sequences may cause issues in our process, resulting in delays in the synthesis. We have designed our proprietary algorithms to check your sequence for potential issues to prevent this from happening as much as possible. Update: due to COVID-19, virtually all DNA suppliers have intermittent delays, which unfortunately affect us too. We keep you updated for each order.

    For off-the-shelf mRNAs, we aim to ship out the same or the next day, so you can get started as soon as possible. However, when you selected shipping on dry ice, we are restricted to sending the material on Monday or Tuesday, so we can be sure it arrives before the weekend and in a frozen state.

    Our turn-around time is affected by the following:

    1. The need for a DNA template. If we need to perform de novo DNA synthesis, 7-10 business days are required to produce the template. If you have a suitable template available, we can start with RNA synthesis 1 day after receiving the material.
    2. Up to 3 days to perform RNA synthesis and the associated QC.
    3. For difficult sequences, troubleshooting may be needed, possibly including DNA prep optimization, RNA synthesis under revised synthesis conditions, optimized purification, re-synthesis to obtain sufficient material, etc.
    4. Shipping to various locations takes up to 3 days. Since mRNA is best send on dry ice, we generally only ship on Mondays/Tuesdays to avoid warm storage over the weekend, possibly resulting in loss of material.
    5. Any special request may add additional time to the order fulfilment. In particular, additional QC or modifications may add extra time.
  • What shipping options do you offer?

    Currently, the consensus is that shipping in frozen state on dry ice is the safest way to transport the fragile RNA. Therefore, we typically send the RNA in syrofoam boxes with 10-15kg of dry ice (depending on destination) with FedEx. The costs for shipment are additional to the price listed because of large variations depending on the destination. We always strive to limit the cost for shipping. Depending on the destination, we only ship dry ice on Monday or Tuesday to ensure arrival before the weekend and in a frozen state.

    To mitigate these costs and reduce turn-around time, RiboPro is currently investigating several alternative shipping solutions; allowing the RNA to be shipped at -20oC., 4oC., or even room temperature. Of course, RNA performance is a key outcome in these tests.

    As a customer, you will always have the choice between aforementioned alternative shipping options and dry ice shipping.

Questions about ordering

  • My preferred payment option is not available, what to do?

    RiboPro aims to be the most customer-oriented RNA provider in the world, as such we are always happy to work with you to enable alternative payment options. Please contact our sales team and they will work out the details with you.

    We offer 2 ways to do this:

    1) You may contact your sales representative by email. He or she will get in touch with you as soon as possible.
    2) You can fill in the form below, which will be send to our sales team directly. Someone will contact you as soon as possible.

  • How can I best order custom mRNA at RiboPro?

    We offer several ways to order custom mRNA at RiboPro and you may choose the one that works best for you:

    1. You may order directly via our website. The RNAssembler™ guides you through all the steps and options to design your own mRNA. Not sure about the end-result? Request one of our RiboProfessionals™ to have a look and if needed discuss with you further.
    2. You may contact our RiboProfessionals™ to help you design your mRNA from scratch, taking into account all relevant parameters of your experiment or application.

    In either case, after you order, you will receive an order confirmation stating all the details you may need. Based on the official quotation that you may generate via the website or via your RiboProfessional™, you may arrange a Purchase order at your institution or company to arrange payment. Alternatively, you may pay via the website directly, or discuss with your RiboProfessional™ alternative payment options.

Other questions

  • I am writing a paper, and can you to check/help with the RNA-related details?

  • I am writing a paper, how should I cite your product?

    If you used an off-the-shelf mRNA, you can simply refer to it by catalog number. You will find this on the product page, but also on your invoice, packing list and product info sheet. The reference could look like: “OTS eGFP mRNA standard (RiboPro, the Netherlands, #catalog no.).

    If you used a custom synthesized mRNA, you can refer to the gene name and sequence of the mRNA, followed by: (custom synthesized by RiboPro, the Netherlands, followed by relevant parameters, including Cap-type, A-tail length, etc.).

    In addition, it is best to include a supplementary figure that includes RiboPro’s quality control and synthesis details. We aim to provide a template for this soon. This template will be downloadable from your account and has the info on your mRNA already filled in.

  • I ran my RNA on agarose gel and it looks smeared. Why?

    RiboPro performs all necessary QC to guarantee identity and an intact product, therefore it is unnecessary to perform such a gel yourself from a QC point of view. Nonetheless, if you run a gel for another reason, you may encounter smearing.

    Smearing of RNA on an agarose gel may have several reasons.

    1. Not using a denaturing gel. RNA fold back on itself by default and can form complicated secondary structures. If multiple secondary structures are present (as is often the case) they run differently on an agarose gel, unless a denaturing agent is used. Use bleach (1% vol/vol), formaldehyde or another denaturing agent in gel and during sample preparation. RNA denaturing requires heating for a short time in a metal/ion-free buffer containing the denaturing agent.
    2. RNAse activity. All RiboPro products are certified RNAse-free. However, from the moment of opening the tube, RNAses may enter the product and may start destruction of the RNA. This also happens, albeit slower at low temperatures and even in a frozen state. An additional source of RNAse is the agarose gel itself.
    3. Heating the RNA above 55ºC with (trace) metals and ions present in the solution. In the presence of common metals and ions like magnesium, RNA is very unstable at elevated temperatures. If your procedure requires heating the RNA, do so in the presence of EDTA and under buffered conditions.
    4. When enzymatic A-tailing is used, the RNA frequently smears slightly due to the variation in A-tail length that is added to each individual transcript by the enzyme.