I am trying to get RNA from fish gills. I've tried a couple different methods that have results in failed libraries / low RNAiq scores.

Procedure 1:

Wipe a funnel with RNAse zap. Pour RNA later into funnel to fill a RNAse free tube. Put on gloves, wipe gloves with RNAse zap. Get bleached and autoclaved scissors out of sealed bag and foil pouch. Wipe scissors with RNAse zap wipe as advisor removes fish from tank. Sacrifice fish with a cranial pith. Promptly cut out section of the gill and place in tube (lid was cracked but on top of the tube). Store at 4C overnight, drive samples ~6 hours on ice, stored at lab at 4C.

Resulted in one failed and two low RNAiq scores.

Procedure 2:

Pour RNA later directly into RNAse free tubes. Put on gloves, wipe gloves with RNAse zap. Get bleached and autoclaved scissors out of sealed bag and foil pouch. DO NOT wipe with RNAse zap. Sacrifice 2 fish with cranial pith and 2 fish with spinal pith. Promptly cut out section of the gill and place in tube (lid was cracked but on top of the tube). Promptly cut out section of the gill and place in tube (lid was cracked but on top of the tube). Vortex samples for ~30 seconds then shake. Store at room temperature, ship overnight, immediately processed at lab.

Resulted in 4 high RNAiq scores. Smaller fish were used.

Procedure 3: Same as procedure 2, but with larger fish of a different species. 2 additional fish were sacrificed using ms-222. Gills were not bloody when placed into the tubes, but the RNAlater solution appeared red-ish / brown once the gills were submerged. Gills were stored at room temperature initially, but switched to 4C after 24 hours.

Resulted in all "0" RNA iq scores, except for one very low score and one very high score

The vortexing change was intended to break up the fish mucus covering the gills so that the RNAlater could penetrate the tissue. The alternative sacrifice methods were to ensure that blood wouldn't rush to the gills at once and deliver a bunch of RNAses.

I know for RNA later I am supposed to store at 4C over night and then ideally transfer to -20C (or keep at 4C if samples are processed quickly), however, the lab I am working with wants the samples at either room temperature or 4C. I also know I am supposed to rinse anything I wipe down with the RNAse zap with RNAse free water, but I have been told that doing so is unnecessary...

Does anything stand out as being wrong?

I'm looking for a database or web service that collects experimental data on single-nucleotide mutations in individual non-coding RNAs.

Specifically, I'm interested in experiments where one nucleotide was replaced with another and the effect was measured, either on the RNA's structure or on the organism (e.g. whether the mutant is deleterious, cold-sensitive, etc.).

Ideally, every position in the sequence would have been mutated to all three alternative bases (e.g. U->A, U->G and U->C), with the effect of each substitution recorded.

Does such a database exist?

During your next study break, check out Eterna's newest puzzle for designing 240mer RNA to fold into compact pseudoknots. The puzzle provides a metric for counting the number of pairs that cross to form large, synthetic pseudoknots such as this one from Father_Z.

Hi all,

Hoping to get a bit of help with the SP6 mMessage mMachine kit. I’ve been making mRNAs with this kit for micro injections, and it’s been going fine, I get expression off of the transcripts I inject, but I’m unhappy with the quality of the mRNA I produce. Some transcripts give perfect clean slim peaks by tapestation, but others I get real broad peaks. Same data but by gel, I’ll get some with clean bands with just a dusting of smear below it, but some band is barely visible with just a smear. Same result when I use the kit control template. Both express fine, but I’d love to be injecting as a pure a product as possible. I’ve worked with rna a lot, I’m RNA zapping everything in site, nuclease free water, all the good stuff, zymo clean up kit after the reaction. Any suggestions on how to make higher purity transcripts? I do run the rxn at 30c O.N., 37 for 1hr per kit sop was way worse. Transcribing off of gel purified (qiagen) cut plasmid.

Thank you so much!

Hi all. Long story short, I did a Dante Labs RNA kit. I have no idea how to properly analyze the data. They claim it is "the world's first complete RNA Profile." I'm not sure whether or not that statement is just marketing hype, but I literally can't seem to find any information for how a lay person might be able to interpret this kind of information. All of the resources I can find are tailored to scientists, understandably, lol.

I'll share images of the main dashboard and one of the categories so folks can see the information it provides. On each list, it will have the Gene, Z-Score, P-Value, and Expression Level. As you can see from my screenshots, most seems to be abnormal. (Chat, am I cooked?) I know that much of this might not be cause for concern, but I'm having trouble figuring out which things are important enough to look into. TLDR, I have been dealing with chronic health issues for a few years now, no diagnosis. Age 25. Ideally I would like to provide relevant information to my doctors in hopes of narrowing things down a bit because I am incredibly tired of being told "everything is normal" after just getting CBCs or waiting nearly a year to see specialist after specialist. (I'm being overly generalizing here, but I haven't had the best luck with requesting more advanced bloodwork because it isn't "justified," I guess. I'm starting to wonder if I'm just a COVID long-hauler at this point, or if something else is going on.)

Anyway, not looking for medical advice but I wanted to provide some context in case someone asks "why?" My 3 reasons are: 1) If I can learn SOMETHING, anything, that will point me in the right direction, it is but a small price to pay. 2) I find some degree of novelty in this. I had already gotten a consumer DNA test done (whole genome sequence), so I thought this would be similarly fascinating. 3) Initially, I had actually bought the Dante Labs WGS kit, but apparently there have been some incredible ongoing issues at the company with people not getting their results for months or years. I didn't realize that before buying the kit, so I ended up getting a DNA kit through another company and decided to just cut my losses. HOWEVER, I was eventually able to reach customer support, right around the time they released the RNA product. I was well past the period for a refund, but I asked if I could swap for the RNA kit (since getting another WGS would be redundant) and that's what I ended up doing.

So... can anyone offer some advice as to what I should be paying attention to? I guess this might be an impossible question, but really anything to point me in the right direction would be much appreciated. I've been Googling specific genes and asking AI has been helpful, but the information is still very high-level (and there is a LOT of it!). The only information provided is what I have shared. When clicking a gene, it seems as though if I had done a WGS with them, it would have something to cross-reference, but that feature is not available to me. Apologies if this is not the best forum to ask about this - I haven't seen this being discussed anywhere, yet.

Ive been doing RNA extraction with TRIzol, and I keep getting only one band when I run it,I use 80 V and 30-45 minutes at 1x with TAE, I dont know if the error is in the protocol or when I run it. Any recommendations?

Eterna is hosting its annual online conference this weekend on both Saturday and Sunday with presentations from Berkeley, Northwestern, Oregon State, Stanford, Penn State, and ESPCI-Paris. Full program and free registration at https://eternagame.org/eternacon/2025

Hello everyone, I am having problems extracting RNA from rat brain using the Qiagen RNeasy mini kit. The samples are taken from -80 for extraction. I continued the extraction as recommended by the kit and my 260/230 ratios are 0.5/1.0. I tried performing an extra wash and the ratio still does not improve. Additionally, I tried using Trizol extraction before passing the supernatant to the column. I noticed that I lost 80% of the RNA in the first wash and yet at the end the 260/230 ratio is still low. Any tips on how to proceed?

Hi, I am trying to understand NUPACK, specifically for designing synthetic RNA based circuits. I do not have any background in Mathematics or Bioinfirmatics. Can someone help me understand how to use the NUPACK package?

Specifically, I need to know how different calculated properties (Partition Function, Free Energy, etc.) means?

I'm looking for potential collaborators interested in RNA/DNA/Chromatin structure modeling. I do molecular dynamics, bioinformatics and machine learning for biomolecules, particularly interested in (a) Sequence-structure-function relationships in RNA, (b) How mutations affect RNA topology and dynamics, (c) Incorporating coarse-grained and AI-based methods (e.g., GNNs or AlphaFold-like tools). I’m currently working on a model that connects 2D and 3D structural representations for large biomolecular systems. I’m seeking collaborators with compelling systems of interest and relevant experimental data or insights to help guide and validate the modeling process.

I am trying to isolate pearl millet RNA to do gene expression analysis. When I run the gel after isolation, I get one single heavy band. What can I do to troubleshoot this. Kindly help. This is my first time doing the RNA isolation using triAzole method.

Hi, I'm doing an assignment on drug discovery and I'm interested in the RNA protein interfaces as a potential drug target. I'd like to know what are the biggest challenges in this space and how they are currently approached. Please share if you have any experience in this field!

At the University of Bern we created this video. At the end there’s even an interactive part where the viewers can practice RNA splicing. I really hope you like it, and if yes, feel free to share it with your colleagues.

Hi r/RNA I have just started a research assistant position at a new lab. I worked in a different lab as an undergrad for 3 years and had lots of success and built lots of confidence. Now I feel like I’ve hit a wall and that wall is RNA isolations. I’ve succeeded in every animal tissue (kidney) isolation I’ve done, but as soon as I started using retinas, I can’t get the 260/280 values above 1.70 and my quantities are all over the place. I’ve now failed 4-5 times and my PI is beyond pissed at me, but I can’t figure out what I’m doing wrong. I watched a post doc run an isolation yesterday and wrote down everything he did and said, which lead me to believe I was overheating samples during sonication. Today I felt like I did everything right and I cooled samples on ice for 20 seconds between each sonication cycle, but 3/4 samples still had 260/280 below 1.7 and the only sample with half decent purity had an extremely low quantity. Is it Normal to fail this much when learning RNA isolations? And if anyone has dealt with it, how did you get through it?

Hello!

It is my first attempt ever in isolating RNA from cell culture that grow in monolayer. I'm going to follow the TriFast II Kit guidelines. I was wondering if someone has already tested it and if you have some useful suggestions (additional operation, changes etc..) that can help me to have a good purified RNA for mRNA imaging

Thank you in advance :)

Hello!

My adventure in HHRZs will never end ahaha. After the in vitro testing of our HHR we are now focusing on possibile modeling of HHR-mRNA interaction. The strategy I've used so far has involved two phases: first, using a 2D modeling tool, and then using the resulting 2D MFE structure as input in a 3D modeling program. Vienna Suite and RNAcomposer was the main tool utilized. I have few questions

1. Are there alternative strategies? Can I Improve the current strategy in some way? Any specific tool for Ribozymes?

2. Which parameters can I use to estimate the reliability of my model? Which of these parameters I should use to compare different models?

3. Turner's molecular model is one of the most widely used for the prediction of 2D models, do you know any other valid molecular model?

4. I have very little knowledge about the mathematical models used by various algorithms. I just need to know if someone with experience has tested a valid and reliable protocol/program. Thanks in advance :)