Questions about products
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.
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 process that prevents dsRNA from contaminating your desired ssRNA product.
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. RiboPro uses 2 methods of cap addition to the mRNA:
1) Co-transcriptional capping; where an anti-reverse cap analog (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.
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 is not affecting the yield and can result in >95% of transcript being capped.
It is worth to note that uncapped transcripts posses 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.
RiboPro has developed a proprietary method that quantifies the capping efficiency. At the moment RiboPro is the only company that offers capping efficiency determination as part of the standard package, even for research-sized quantities. We do so because of the enormous impact of capping on mRNA performance.
To keep our competitive advantage, we do not disclose the method. What we can tell you is that our method is between 1-5% accurate, meaning we specify the capping efficiency in brackets of 5%.
Currently, we offer 2 methods of adding the A-tail to the RNA:
- Enzymatic addition. The length of the A-tail is determined by the reaction time. This method produces a Gaussian distribution of A-tail lengths around the desired length.
- 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.
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 catalog 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 that is added by a denaturing agarose gel.
Smearing of RNA on an agarose gel may have several reasons.
- 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.
- 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.
- 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.
- 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.
Questions about the process
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.
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.
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 dry shipping solutions; allowing the RNA to be shipped in a dry state at room temperature. Of course, RNA performance is a key outcome in these tests. If succesful, RiboPro will be offering dry shipping at room temperature from the 1st of March 2020.
As a customer, you will always have the choice between dry shipping and dry ice shipping.
Questions about ordering
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.
First of all, our apologies for the inconvenience this causes you. We are committed to solve this as soon as possible.
In the mean time, may we ask your help with the following 2 things?
- Please process your order by clicking the option “process order and contact me about payment”. This way, you do not have to wait longer than necessary. If you are a returning customer or if you have a small order, we will start your synthesis immediately. The order will be shipped once payment has been arranged. If you are a new customer and have a large order, we may contact you to get payment sorted before we start synthesis.
- Please fill in the form below, so we can solve the problem as soon as possible?
Many thanks in advance.
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.
At RiboPro, we are invested in your success and thus. we like to help you as much as we can. Depending on the extend of the work involved, we can help you for free or require a small fee.
Free: up to 3 sentences related to RNA checked in the main body of your paper.
Free: check-up of the RNA details in your materials and methods.
Paid: more than 3 sentences related to RNA in the main body of your paper.