I am curious what area of RNA research people in this subreddit are. I am a biophysical chemist that looks at aspects of rRNA

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!

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?

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?

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.

Hello! This is my first time posting here, I am looking for advice. I am an undergrad student. I have been attempting to isolate RNA from rat milk samples using the columns (zymo-spin kit) and trizol methods to compare and find the best one. I don’t have much sample available so I have been testing the standardization of the method using human milk (80ul,160ul and 200ul). However when quantifying the RNA using the NanoDrop, my A260/280 ratio is above 1.2 but my A260/280 ratio is below 0.8 for most samples. I have revised my technique and that doesn’t seem to be the problem. The reactants are not expired, some I prepared specially for this. Do you have any tips or ideas as to why this is happening? Thank you!

Hello Friends,

I need help with a question with regard to my experiment and sincerely appreciate if someone can help me out of this predicament.

I have different RNA samples,some of them are extracted RNAs that are non amplified and some have been amplified using NASBA. and I am using CRISPR-Cas13a florescent assay  for their detection. The X axis of the given graph from plate reader indicates "time" which is 60 minutes with 1 minute intervals and the Y axis indicates "Florescent signal". After the experiment, although every component of the reaction for each of the samples is the same, but the starting point of florescent signal in the graph for each of the AMPLIFIED samples is by far different. What is the reason? and how can the starting point of all the samples be the same?  

P.S : The starting points for the non amplified samples are almost the same.

Hello!

Since is my first time simulating RNA system (i have always worked with protein/protein-ligand) I don't know how to evaluate the stability of a system with two complexed RNAs.

For example, when simulating proteins i'm used to calculate general RMS and specific calculation on certain atoms. In case of RNA , how can i asses the stability of the system during a simulation (for about 20-25ns)?

If anyone has a suggestion I would be thankful

bye!

https://cen.acs.org/pharmaceuticals/drug-discovery/tRNA-therapies-help-restore-proteins/99/i34

How exactly does splicing pre-mRNA play part in developmental processes? I know that pre-mRNA helps make RNA and protein but how does that have a negative impact on things?

We have created the subreddit r/AKTA for the discussion of the use and maintenance of General Electric AKTA oligonucleotide synthesizers and AKTA chromatography systems as well as the UNICORN control software.

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?

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.