RNA - Science topic

I have been trying to isolate RNA from magur fish testis, at the end of RNA isolation, RNA pellets becoming gel when DEPC water is added, what could be the possible reason for that? What is the solution?

Does anyone have any advice regarding 16s metagenomics and an appropriate sequencing depth and paired end reads. Expecting around 10-15 organisms within the samples.

Thank you :)

For researchers who have conducted transcriptomics using the adult rat prefrontal cortex, is it necessary to optimize the RNA extraction method? Additionally, what is the average weight of the rat prefrontal cortex, and how much RNA can typically be extracted from it?

Who (first) proposed/used/coined the term ‘translation’ in biology/genetics? What is the history behind the use of the word? Thank you!

The amount of total RNA used by each group was different during reverse transcription. Can it be compensated by adjusting the same CT value during PCR?

Unfortunately, there is a delay of about an hour between the the tumor extraction by the surgeon and sample freezing. How can I protect the RNA in the tumor from degradation at this time?

Hi,

I recently made a mutant cell line by excising an exon from the gene, DNMT1, using CRISPR-CAS9. I isolated a single cell population that has this mutation and confirmed the mutation using PCR and Sanger sequencing. My PI also wants to use RT-qPCR to show that the sequence is missing in the mRNA. I made 3 sets of primers targetting the exon, so I would only expect amplification in the negative control cell line and not in the mutant line. However, when I ran the qPCR, I got normal amplification of this DNMT1 exon in both the negative control and the mutant line (~ct values around 23 for both).

I've extracted RNA three separate times to make sure I didnt have RNA contamination the times prior, but I still get the same result.

If anyone has experience with this or may have solutions, any help is appreciated!

Hi all

earlier I have seen in some papers people go for DNA extraction and normal PCR using 16S rRNA primers for the identification of bacteria. However recently I have seen few papers particularly dealing with Uncultured “Candidatus” bacteria, researchers go for RNA extraction, reverse transcription RT-PCR and real-time RT-PCR ? Molecular biology experts can you please tell me …..

1. what’s the key advantage between the two ? is there any particular advantage of RT PCR for the identification of Uncultured “Candidatus” bacteria ?

2. Is it because of the possibility of “relative quantification” of the bacterium by real-time RT-PCR by targeting the 16S rRNA gene of the bacterium?

3. Is there any advantage when (RT PCR) used for uncultivable bacteria?

4. what is this Cycle threshold ? what is the significance of this in the above reaction ?

5. Also “The eukaryotic elongation factor 1 alpha from the host was used as a control of the RNA amount, and a good extraction was expected to give a Ct-value around 15 (the cycle threshold was set to 0.1). ? all results with Ct-values above 45 were considered negative !, what does it all mean?

My aim is just to identify the unculturable bacteria from tissues! Can I go for just normal PCR (16s rDNA) and sequencing the PCR products? Please

thank you

regards,

Hello everyone,

I have performed EMSA experiment with an RNA-binding protein and RNA. For both ssRNA and dsRNA, for higher concentration of protein, I observe a high intensity band at the dye front (lower than the free RNA band). The intensity of this band decreases with decrease in protein concentration. I have performed the experiments in a cold room with 0.5X TAE buffer and 8% gel.

Any idea why this is happening?

I'm teaching a student to do Drosophila embryo whole mount RNA in situs. She's using the same solutions that I use however her embryos turn light tan to medium brown after incubating O/N in prehyb. Does anyone have suggestions for how this is happening?

Prior to the research carried out by the research group who published this journal, they assessed HDV RNA detection's diagnostic efficacy utilizing the HDV RNA detection technique and discovered that it had a good diagnostic yield. However, HDV serology's diagnostic effectiveness hasn't been thoroughly assessed and documented, thus, the need to consider the factors needed in developing a reliable serological test for HDV antibodies.

So I'm using TRIzol for RNA extraction, but suddenly I'm getting no pellet during the isopropanol step *even though I added glycogen.*

A few weeks ago I:

1. Took a large quantity of bacteria

2. Resuspended in TRIzol

3. Made a bunch of aliquots

4. Stored them all at -80

And those couple weeks ago, I was getting~50 ug of RNA per aliquot (with good RINs, and they looked great on RNA gels).

Yesterday, I took another aliquot and tried to prep it using the exact same process, and got nothing. No pellet appeared during the isopropanol precipitation step even though I added GlycoBlue.

I continued the purification to see if the pellet was just hard to see: ~0 ng/uL by nanodrop

Did I just make a pipetting error? Was my isopropanol bad? No: I repeated the repeated the process *again* with completely new sample, completely new isopropanol, and still no pellet at all.

I'm confident I'm lysing my samples well. I optimized the lysis a while ago, but even before optimization, my yields were never this bad.

I'm sure I added the GlycoBlue. I watched it diffuse into my sample.

I'm sure I added isopropanol. I watched the alcohol/water mix and used brand new isopropanol.

I'm sure I mixed the isopropanol/aqueous phase. I watched it carefully.

I'm sure I actually spun my samples. I tried it over and over.

Protocol:

1. Take bacteria+TRIzol aliquot from -80 (which used to give ~50 ug)

2. Lyse via bead beating (same beads, same bead beater, same settings, same duration that gave 50 ug in the past)

3. Add 0.2 V chloroform

4. Centrifuge 12k xg for 15 min

5. Take upper, colorless aqueous phase

6. Add 1 V of RT isopropanol

7. Add ~30 ug GlycoBlue

8. Invert a bunch of times to mix well

9. Incubate 10 min RT

10. Centrifuge ~20k xg for 10 min

Nothing. No pellet at all. Even with glycogen.

I tried spinning again: No pellet

How is this possible?

Hi,

I have recently been working on RNA purification from RNA-binding proteins. I used a protein-specific antibody to immunoprecipitate the protein RNA complex and then extracted RNA from the complex. I used Trizol to extract RNA and precipitated the RNA using ethanol. After centrifugation, I got a liquid drop of RNA but not a solid pellet.

Does anybody have any idea about what happened?

Thanks

Baoye

I am about to perform 5'RACE with TAKARA kit on total RNA isolated (trizol method) from neuronal samples differentiated in the same time frame. So I have 3 RNA replicates of one sample, all isolated at the same time. Can I combine the RNA (for example ,1 microgram from each replicate) from replicates of one biological sample and then perform the 5'RACE ?

Dear Researchers,

I have a couple of falcons with collected media from cell cultures. I have been storing them for a year in 4*C. They were collected for an experiment that I hadn't had the chance to finish. My question is, can I perform any analysis on them while they were stored in such conditions for this long? Anything with DNA/RNA/proteins? Or everything is degraded by now? I will appreciate every idea.

I wanted to add a methyl group in the adenine N6 position in the RNA for some docking and simulation studies. Is there any protocol or CODE for such editing to CH3 to the existing RNA ?? Is it possible in the discovery studio or modeler??? what is the process??

#INSILICO_BIOLOGY #Computational_Biology #DISCOVERY?_STUDIO

Hello All,

Do you have any suggestions to extract RNA from frozen blood samples collected from mice, for miRNA studies? The blood samples were stored in Eppendorf tubes at -80C.

Thank you!

what is an acceptable RIN level for an RNA sequ experiment? G

Hi,

I am growing Caco2 cells in 24 Transwell cell inserts. I am planning to induce gut leakage by LPS and want to study gene expressions. My concern is to isolate the RNA from transwell inserts since those are very tiny and hard to use cell scrapper. Any protocol or suggestions would be greatly appreciated

I am planning to get some blood from patients to perform RNA seq (circular RNA which requires deep sequencing). I am wondering if anyone can share the protocol with details. I am also wondering about the amount of blood (whole / plasma / serum) to isolate RNA for deep sequencing. Thank you so much for your help!

I'm getting very low yields - 1/2ng and unfortunately can only take 20mls from the donors. Any help would be much appreciated!

Hey,

Usually, when I perform total RNA extraction, I directly synthesize cDNA and run qPCR without even analyzing or testing the purity of RNA. I am asked to use 15µl of RNA when synthesizing DNA. However, I am not able to understand the reason that is given to me, because in academia they taught us that RNA has to be tested for purity before proceeding with further experiments. I also don't understand the idea behind just using 15µl exactly to synthesize cDNA instead of calculating the exact amount needed. If there is logic behind that, can someone explain it to me in simple words?

Thank you

Hello everyone. I am interested in performing a time-course bulk RNA-seq experiment of FACS sorted cell populations. I will use a live-cell fluorescent for sorting so fixing before sorting is out of the question. I need to sort cells at 0-6h-12h-24h-48h-72h but I want to prepare the RNAs at the same time from each sample. Is it possible to fix the cells after sorting and isolate RNA later? Or is it possible to freeze the cell pellet after sorting and isolate RNA later? Any recommendations by any means (protocol, kits, etc.)?

I've been trying to extract RNA from mouse lung tissues (normal and tumour) and slowly improving the yield and purity measured via Nanodrop and Qubit (I'm consistently getting 260/280 ratios of ~2 and 260/230 >2 + yields of 40-100 ng/uL). I'm using the Qiagen Mini kit with DNase I and have been using all the small tweaks I can find from here and papers to optimize my yield. I've also been trying to work with RNaseZap and maintain as clean of an environment as possible.

My trouble currently is with RNA degradation- on the Bioanalyzer my samples have come back with RIN values with a range of 5.4 to 6.9 (attached), which from what I've read is unfortunately too low for RNA-seq. I've been using an OMNI tissue homogenizer for 20 seconds x3, is this possibly leading to shearing of my RNA? Has anyone else used a rotor homogenizer on tissue and had good RIN values? I can't think of what else to optimize and I'm wondering if it's potentially a sample issue (the samples are 2-4 years old stored in the -80), but I want to maximize everything I can before coming to that conclusion.

Would another extraction method potentially lead to less degradation? Would Trizol + then running it through an RNeasy column potentially help with this issue?

I want to covert RNA aptamer to DNA aptamer.

I try this process by Discovery studio.

This software includes the "Build and Edit Nucleic Acid tool".

I could modify the sugar easily.

However, I edit the 5H to methyl in U to convert T with manually process. It is very time-consuming.

Do you have a good idea to convert U to T easily?

Hello!

I'm extracting RNA from fiddler crab (Leptuca pugilator) larvae and embryos for Tag-Seq. I've extracted using an RNAqueous RNA Extraction Kit and we wanted to determine RNA quality and integrity using the Agilent 2100 Bioanalyzer System.

I'm attaching screen grabs of the gel and peaks produced by the bioanalyzer. I expected to see 28S and 18S bands and peaks. However, I have one larger band on the gel and one large peak that seems to lie between 28S and 18S.

Has anyone seen anything similar using this instrument? Any insight would be highly appreciated. I would love to hear from someone that's worked with a similar species if y'all are out there!

Hi All,

I would like to seek for your expert input for below queries on RNA QC for RNASeq:

Can we proceed for RNASeq run on RNA less than 10ng, with DV₂₀₀ in between 68 - 80%?Details as below:

Sample 1 Sample 2 Sample 3 Sample 4 Sample 5 Sample 6

ng/uL 4.36 5.36 4.14 4.69 2.85 14.7

A260/280 2.472 2.061 1.636 2.057 1.968 1.968

A260/230 2.044 1.511 0.857 0.837 0.610 2.011

RQS NA NA NA NA NA 2.6

DV₂₀₀ 68.9 80.3 75.5 72.2 80.5 71.8

FYI, the RNA samples were extracted from the laser capture microdissected tissues.

Many thanks in advance!

For DNA islolation

My student optimizes the RNA dot-blot method using a biotinylated cDNA probe. The aim is to detect a positive hybridization signal of a probe complementary to transcripts of a housekeeping gene for Human actin beta in the human RNA samples. So far, we have not been able to detect consistent hybridization signals. Either there were non-specific, very weak or no hybridization signals at all. Above it, we have recently observed a reverse white signal in dot-blot spots with our human RNA after colourimetric detection. The only such phenomenon I found when googling for the solution appears after chemiluminescent signal detection. The alleged ghost bands or spots appear when a sample or antibody excess consumes chemiluminescent substrates too quickly. However, in the case of colourimetric detection, the chromogenic substrate accumulates instead of being consumed, even in the excess of both sample and antibody. In search of a solution, we performed a dot-blot with a matrix of sample and antibody amount combinations, which resulted in either reverse or no signal et all. Does anybody have any idea where the problem could be?

Please, see the attached photo of the dot-blot results. There are 3 membrane strips, the numbers 1, 3, and 5 at each strip indicate the antibody dilution 1:1000, 1:3000, and 1:5000 respectively. The pink strip on the top of the photo shows the identity (and in the case of RNA samples also the total amount) of loaded samples in the indicated order. There are total RNA samples at the concentrations 5 ug, 500 ng, and 50 ng, followed by positive control spots of biotinylated lectin (positive control of colourimetric development of a signal produced by reporter molecule HRP in conjugate with streptavidin) and the rightmost spot is the labelled probe.

I will be very grateful for any suggestions!

I am working on a variety of DNA and RNA structures (generally quite short 20-40bp) and I have seen some worrying results around stability. I understand the nature of the solution will affect the structures ie ice crystallization, pH etc but I was just wondering what others have used and why? Obviously there's Glycerol, trehalose etc any off the shelf solutions others can recommend before I spend the money?

Thanks

I would like help reading the quality and integrity of my RNA. This picture makes me think all my RNA seems degraded?

I have been trying to extract RNA from mouse lung tumor and normal tissue for the past month with varied success in terms of concentrations and purity from Nanodrop and Qubit (concentrations too low to measure to 80 ng/uL). My tissue sizes are very small (about 7 to 20mm3), so I've been trying to do all I can to maximize my yield like using RNALater-ICE and using RNAse Zap on my equipment. I currently use an Omni tissue homogenizer for 40s on my tissue in RLT buffer plus beta-mercaptoethanol. My extractions have been done with the Qiagen Mini kit and I've read all the posts I can find on here and several papers about how to optimize RNA yield with this kit; yet my yields are just averaging about 40 ng/uL of RNA.

I've decided to add the optional DNAse digestion step to my extractions and I wanted to check for gDNA contamination and assess the RNA quality of my extractions by gel electrophoresis since we do not have access to a Bioanalyzer. I've seen that RNA can be run on native gels, so these pictures are of a 1% agarose gel with 1X TBE and 60V at 60 minutes with a DNA ladder to check running of the gel and my RNA samples +/- DNase, samples were mixed with 6x loading dye. Are these images indicative of RNA degradation or do I need to run a non-denaturing gel (if so, how do I do that)? There's a dark pinkish band on the bottom half of the gel that's hard to see in the picture very clear in normal lighting and I'm not sure what that represents? I definitely don't see bands for 28S and 16S so I'm feeling kind of hopeless that all my RNA quantities measured by Qubit represent poor quality RNA. I would like to send my RNA for RNA sequencing eventually (not specifically from these samples, which are more for practice).

Thanks so much in advance for reading through and offering any guidance.

I'm using TRIzol+bead beating to extract RNA from a gram positive bacteria, but I'm getting very low yields (<1 ug total). Years ago, people in my lab got more like 50 ug RNA from this same quantity of bacteria, and I calculated a theoretical max yield of ~200 ug.

I take 2-6*10^9 cfu

Wash once in PBS

Resuspend in 1 mL TRIzol

Bead beat

Then I follow the exact protocol described in the TRIzol documentation:

https://assets.thermofisher.com/TFS-Assets/LSG/manuals/trizol_reagent.pdf

Starting with step 2 for "Lyse samples and separate phases"

Which is just a generic alcohol precipitation.

The protocol is basically shown here:

http://www.dartmouth.edu/~staphy/protocol/northern_rna_extract.html

I'm not losing the pellet as I have GlycoBlue and I can see a tiny, tiny, tiny blue pellet that I can resuspend.

As far as I understand, this can't be degradation as degraded RNA will still pellet. Is that correct?

Therefore, it must be incomplete lysis. Is that correct?

But I've checked the literature and found papers using the exact same bead beater I use. They all use the same or shorter bead beating times.

http://www.dartmouth.edu/~staphy/protocol/northern_rna_extract.html

This protocol uses the exact same bead beater and lysis steps: 3 min total with a mini-beadbeater-8

Am I correct that degraded RNA will still precipitate? How could my lysis be incomplete if it works for everyone else?

I am doing western blots with an RNA binding protein and believe due to the vertical streaks, that I have RNA contamination. I have tried boiling the samples for longer as well. Another protein I have probed for does not have the vertical streaks and is not an RNA binding protein. Could someone recommend a protocol they have used or know of to get rid of RNA contamination in protein preps for western blots?

Various pandemic diseases have taught us various lessons from time to time, lastly, the spread of corona virus spread has shown how fickle human condition or survival is in face of sudden outbreak of dangerous diseases!

What are the human security implications of 'corona virus spread' around the world?

I wanted to confirm the binding of a lncRNA to a known protein. The protein is mostly disordered except for the part that may be involved in the binding (a coiled-coil region at the end). I was thinking of performing an EMSA to confirm the binding. And then maybe I was thinking of doing a CLIP-seq to delve deep into the binding motifs and sites to see if the RNA binds to the binding domain or some other site of the protein.

My question is - is CLIP-seq something that one can perform after establishing a binding between two components? As far as I know, CLIP-seq can give more detailed and confirmed binding data concerning motif and site-based proof of the RNA binding to the protein. If not, what can be some other in-vivo experiment that can be done to check the nucleotide-level binding of the RNA-protein?

Greetings!

I started my thesis and I need to research on the methods to extract RNA from vine leaves.

I am facing an issue regarding the concentration and purity of RNA, the 260/280 ratio shows contamination.

1. Aspirate media from plated cells.

2. Rinse cell monolayer with room temp PBS only once.

3. Scrape the cell monolayer with cell scraper.

4. Transfer cell to 1.5 ml microfuge tube. Centrifuge at 5000 rpm for 5 min.

5. Remove the supernatant and added 400µl TRIzol. Mix till the pellet dissolve by pipetting thoroughly.

The volume of TRIzol added should be as follows:

For 1 well of a 6 well plate or a 35 mm plate use 400 µl TRIzol.

(After this The lysate is stored at -80 degrees C).

6. Before the phase separation the lysates are thawed on ice.

7. Incubate the lysate for 5 minutes at room temperature.

8. Phase separation: Add 0.2 ml of chloroform to the supernatant (per 1 ml of TRIzol reagent).

9. Shake vigorously for 15 seconds and incubate at room temp for 2-3 min.

10. Centrifuge for 15 min at 12,000 x g at 4ºC.

11. Following centrifugation, the mixture separates into lower red, phenol chloroform phase, an interphase, and a colorless upper aqueous phase. RNA remains exclusively in the aqueous phase. Transfer upper aqueous phase carefully without disturbing the interphase into fresh tube.

12. RNA precipitation: Precipitate the RNA from the aqueous phase by mixing with ice cold isopropyl alcohol. Use 0.5 ml of ice cold isopropyl alcohol (per 1 ml of TRIzol reagent used). Incubate for 10 min on ice (if cloudy, precipitate for an additional 10–15 min).

13. Centrifuge at no more than 12,000 x g for 15 minutes at 4 degree C.

14. After centrifugation, a small white RNA precipitate should be visible on side of the tube at this point.

15. RNA wash: Remove the supernatant completely. Wash the RNA pellet once with 75% ethanol, adding at least 1 ml of 75% ethanol (per 1 ml of TRIzol reagent used). Mix by flicking and inverting tube.

16. Centrifuge at no more than 7,500 x g for 5 minutes at 4 degree C.

17. Repeat the above washing procedure once. Remove all leftover ethanol.

18. Air-dry RNA pellet for 5-10 minutes.

19. Resuspend the pellet in 20ul RNase free water.

20. incubate at 55ºC for 3 min.

21. stored at -20 degree C.

The nanodrop reading is very low (in tens and hundreds) and the 260/280 ratio is coming in 1.8-1.9 range. What is the issue I am not able to understand. Is it due to a handling error or do I need to change in protocol something?

Is there any reason for i failed getting any bands on electrophoresis after i did plasmid isolation from E. coli DH5a? My plasmid is ~17 kb in size and i already checked the transformant with colony PCR but strangely i did not get any plasmid band after i isolated them using alkali lysis method even i have confirmed the pellet presence.

I already include RNase A in my isolation protocol so i am quite sure that the pellet was not from RNA. I also have tried to upscale the culture volume to 50 ml and did the midiprep version of alkali lysis. I already reduced the elution volume to 30 ul. I even tried to use Presto Mini Plasmid Kit from Geneaid but i got no result. This is the first time i encounter such problem. If anyone could give me some suggestions i will be very glad.

p.s. currently i don't have access to nanodrop because the facility that has it currently on lockdown

Hi guys, I am currently working on qRT-PCR. However, the amount cDNA I got from RT reaction is too little to assess all the targets.

I saw some papers and websites mentioning amplifying cDNA with PCR, I wonder if it is a good thing to do, or if it is better to redo all from the very beginning (extract RNA from tissue).

Many Thanks!

Hi! I am trying to do RNA extraction from p7 mice's somatosensory cortex and hippocampus. But I am not sure if the RNA was extracted from the correct brain region. I was wondering if there are any standard ways to test it. Thank you.

Hello, I am using the conrad chromatin-associated RNA extraction protocol.

Everything was fine, until it was time to resuspend the pellet corresponding to the chromatin fraction, since a kind of jelly was formed, which absorbed the water.

I need to know how to solve that and if it is normal.

How can I solubilize the pellet?

What is the most effective kit or method for extracting RNA from a very small number of cells?

I've been conducting RNA extraction from 200 microliters of whole blood using the Purelink RNA Minikit according to the provided instructions. Despite several attempts, I'm consistently obtaining low yields (less than 20ng) with poor quality, indicated by a 260/280 ratio of 3+ and low 260/230 ratios. Unfortunately, I'm unable to switch to a different kit at the moment. Could someone please give me some advice on how to improve both the yield and quality of my RNA extraction?

Hi,

I want to study RNA interacting with my test compound

I am planning to do a RNA- pulldown experiment using streptavidin magnetic beads and biotin conjugated compound. I will detect interacting RNA by PCR.

I am wondering how to elute the RNAs from beads, heating may degrade the RNAs.

Any suggestions appreciated.

Thank you

ratio is around 1.4-1.8

Hi fellows, I've started a project in a new lab where I have to extract RNA from mice liver, brain, and muscles. Upon dissection I immediately snap-freeze the tissue in liquid nitrogen and store it for later isolation. When I had to isolate, I separated a piece of the tissue frozen in the tube with small tweezers, put it in trizol, and returned the tube to -80C.

I've been reading some places and it occured to me that the tissue must be ground to powder while it's frozen before it's used for RNA isolation in trizol. Can anyone please clarify this procedure (everything you do with the dissected tissue up until you put your sample in trizol and homogenize it)? Why is the tissue ground to powder? How long do you wait after snap freezing your tissue to grind it? What tools do you use? What do you do to make sure you preserve the RNA in your sample? How do you measure 50-100mg of tissue (Trizol protocol says thats the range u should use in 1mL) while avoiding the thawing of the tissue and activation of endogenous RNAses?

Any insight will be appreciated.

Please send me some standardized manual protocols for the extraction of RNA from Streptomyces sp.

Thank you in advance.

Hello Fellow researchers !

I am doing RLM 5'RACE to validate mirRNA cleavage on it's target genes using GeneRacer kit (Thermo Fisher Scientific). After when purified mRNA using Dynabeads where i used ~32ug of RNA and yielded 17ng/ul for 20ul of volume. After searching i found it is potentially more than 1% of of my original RNA (~32) which i moved ahead with ligation step. I followed this link from page 22 https://www.thermofisher.com/document-connect/document-connect.html?url=https://assets.thermofisher.com/TFS-Assets%2FLSG%2Fmanuals%2Fgeneracer_man.pdf and once i finished i ran 1ug of ligated mRNA with 1 ul of 6X loading dye and 2ul of water. I don't see anything on the gel except my ladder, is it normal? I was wondering if SYBR stain is good to stain mRNA or since this time i diluted mRNA to run on the gel and it was pretty less amount of mRNA which is why i did not see anything.

it would be really helpful to get your comments :)

Thank you

Using a standard rt qPCR kit. When looking at non human miRNA species there is a sort of 'background level' of expression in my results when there shouldnt be - i have tried running samples of pbs, Te buffer, RNA storage solution, H20 as a negative control and they all express at around 33 Cts - i have tried opening new RT kit, new qPCR master mix, new assays/primers, new pipettes, changed all equipment, completing in a pcr hood, all new reagents and a different lab ensirely. I use bleach, RNAseaway and ethanol all the time to remove any contamination. Still no change. I dont believe its from contamination at this stage because of how 'stable' every repeat is - I tried a stabiliser also and that is always clear too, which is also evidence it is not contamination going on here. Any sort of buffer seems to express at ct 33 has any one else experienced this? Do i have my threshold wrong on my qPCR machine? is there something going on with the sequence binding? Please help ! any advice appreciated.

Has anyone extracted total RNA from stained H&E slides? We have cases in our study that have no tissue left in the FFPE blocks, and no other tumor source.

Hi all,

I have been trying to synthesize luciferase self-amplifying RNA (saRNA) by in vitro transcription (IVT), and this vector has approximately 11kb. The main problem is that I am getting a product of approximately 8kb. (p.s. I am using the NEB HiScribe kit)

I have linearised my plasmid with MluI and the bands on the gel seem to be fine (see the picture attached). Upon completing my RNA IVT, I usually get a good RNA yield that is around 800-1000 ng/ul.

The main problem seems to be the premature termination of the polymerase, hence the shorter RNA product. Does anyone know if there could be other causes for this? Or how to prevent premature termination?

Many thanks,

Beatriz

I encounter the following problem: I want to send viral RNA for further analysis. As the receiver requires, I must process my samples according to a regular RNA extraction protocol and send the filtered spin columns from the RNA extraction kit, without performing the final elution step. But I don't have the original sample preserved, only the RNA already extracted is stored. Can I do a re-extraction using the stored eluate as the starting material?

Hi everybody,

Im trying to isolate RNA from umbilical cord total tissue by nitrogen freezing and powdering and then Qiazol/Choloroform extraction. I have obtained good results with placental total tissue but with cord the problem is that the pellet is very viscous, and when I try to solubilize it in water to quantify, I cannot even to pippete because of this.

Could anyone show me a protocol to do this?

Thanks

Total RNA concentrations aren't bad, but the PCR never comes out. What can I do?

I have tried isolating RNA from dead roots with no success. We call them dead just because they are discolored and black/dark brown but not sure if that means they are completely dead. I have tried many RNA isolation methods but none works for these particular roots.

Any insights?

I have found some trouble with succesfully extracting RNA from ToBRFV infected tomatoplant samples. Currently I am using the MolGen 96-prep machine. When I put RNA on 1% agarosegel it is all smeared and lowered in the gel.

For cDNA synthesis I am using random primers (100 µM), Maxima reverse transcriptase (1:5) 0,1 µL per sample, RNase free water, RT-buffer, DNTP's (10 µM). I use 2 µL of RNA to synthesis. PCR programme as followed:

25◦C 10 min

65 °C 30 min

4 °C 3 min

50 °C 42 min

85 °C 5 min

12 °C ꚙ

I can use some tips and advice to improve these experiments. Because I am working on this for 6 months now without some useful results.

Thank you in advance.

Hi!

I'm using the TRIsure method to isolate proteins and RNA from tissue (the procedure is similar to Trizol method). I usually store the RNA precipitacion with isopropanol at -20°C overnight for the next day I get the pellet. Could RNA be left in isopropanol more days, like all weekend, without effects on quality? Thanks

Does anyone have any protocols?

Does anyone have a protocol/experience with extracting total protein or RNA from formalin or PFA fixed tissues that have not been paraffin embedded? I want to extract protein/RNA from mouse brains that were fixed by perfusion with 10% neutral buffered formalin. Older samples are cryoprotected in 30% sucrose, but if I can cut a piece of tissue before the sucrose step that is also doable.

I want to perfuse my mice to better preserve tissues for cryosectioning and IHC, but it would be nice to use less animals and perform less surgeries especially since I only need little protein or RNA to run Western blots or RT-qPCR experiments and it seems a shame spend extra time and mice.

I have searched the literature and see many papers discussing techniques for FFPE sections, but no discussion of fixed tissues that are not paraffin embedded or mounted on slides.

I use a DNA/RNA synthesizer from LGC (Biosearch), a MerMade6, to make oligos. The machine has operated for around 16 months and produced about 1500 oligos during this period. As you can see, it is not a heavily used machine.

All preventive maintenances were ok, and the machine was operating just fine and yielding very good quality oligos......

But then it started to present a misalignment between the cart/columns block and the capilaries, pouring reagents out of the columns.

If you look closely you can see that, when the cart with the columns moves and gets to the position assigned, it doesn't stop completely, but slides juuuust a little bit instead. Such a mess!

Calibration doesn't solve the problem: after a calibration, reagents will be delivered correctly into the columns in the first cycle, but as with every move the cart slides a little bit, the misalignment starts to sum up with every move and, at the 3rd cycle, reagents are being spilled outside the columns already.

We are in Brazil. Remotely, LGC team couldn't help. They couldn't even find the problem. The visit of LGC engineering team will coast a lot, and while we are working in resources an bureaucracies to get them here, tips from other users would be welcome and much appreciated.

This machine works with step motor / step motion, which is similar to printers and a lot of other machines. Have you ever seen this kind of issue?

Alternatively, do you know other MerMade users you can put me in touch with?

Thanks in advance

We are slicing our human hippocampus with cryostat and wanted to know how much RNA (in ug) can you obtain from a cryostat slice? Obviously it depends on the thickness, but if anyone has any insight, please share!

Hello RG Hivemind,

For RNAseq of Aspergillus (or other filamentous fungi) with Oxford nanopore MinION, I am wondering:

1. What is the expected percent yield purified mRNA from total RNA? From a cursory search, a range from 1 to 5 % mRNA/total RNA seems standard at a broad taxonomic scale, but what is your experience with fungi?

2. Anyone done PCR-free direct cDNA sequencing with Aspergillus? With low mRNA concentration we may investigate PCR-cDNA sequencing instead.

Some background:

Using liquid N2 mortar-pestle homogenization and Qiagen RNeasy Plant Mini Kit we extracted total RNA from Aspergillus (cultured on liquid media) mycelia with good yield (~300 ng/μl, Qubit IQ integrity = 10).

Using this total RNA as input to the Oxford Nanopore direct cDNA sequencing kit, cDNA synthesis was performed on four input concentrations of total RNA ranging from 0.5 to 5 μg, resulting in a cDNA yield plateauing around 1.75 ng/μl (see attached image; analyzed after RNA degradation and second strand synthesis but before End Prep and Barcode Ligation).

Given that 70 to 200 ng of cDNA are required as input for End Prep and barcode ligation, my cDNA yield is prohibitively low, but perhaps not lower than is expected given the small fraction of mRNA in the input total RNA?

Of course, why not purify the mRNA before cDNA synthesis? Using the NEBNext Poly(A) mRNA Magnetic Isolation Module with 2 μg input total RNA, effectively all mRNA was lost resulting in below 1ng/μl yields of low purity mRNA, if any.

So my choice is between:

(A) troubleshooting mRNA purification until yield is high enough (≥100ng for direct cDNA sequencing or ≥1ng for PCR-cDNA sequencing), or

(B) use ≥50ng total RNA as input for PCR-cDNA sequencing.

I used the NEBNext Poly(A) mRNA Magnetic Isolation Module previously with wasp tissues and had no issue getting good mRNA yield from the insect total RNA. So I am puzzled and open to advice from anyone working in this area :) Thank you in advance for your time and your advice!

- Andrew Legan

Does anyone know which tubes the PAXgene Blood RNA Tube corresponds to? Are they EDTA tubes or Heparin tubes?

Best regards,

Hind Sebbah

Hello, I am currently isolating nuclei from adipose tissue and I am interested in obtaining high-quality RNA for further assays. What extraction methods do you recommend?

I´m currently using the TRIzol protocol, without modifications but in addition to obtaining very little amount of RNA, the ​​280/260 and 280/230 values are very low, any suggestions?

Colleagues, tell me which dye is optimal for staining RNA during gel electrophoresis? Which one do you use in your laboratory?

I am trying to adapt a DNA extraction protocol to allow me to extract both RNA and DNA from the same sample. I plan to put the sample through Qiagen Allprep DNA/RNA mini kit, but I am not sure at what step I lose the RNA.

SDS/CTAB cleanup

a. Add 10 μl SDS (10%) + 1 μl proteinase K (10 mg/ml stock) to each tube and incubate at 56 °C for 20 min. At this point, pre-incubate CTAB/NaCl solution at 65 °C.

b. Add 35 μl NaCl (5 M) + 28.1 μl CTAB/NaCl (2.5%). Pulse vortex. Incubate at 65 °C for 10 min then perform a quick spin.

c. Add 200 μl phenol:chloroform:isoamyl alcohol (25:24:1) pH 8.0. Pulse vortex. Centrifuge 8,000 × g for 5 min at RT.

d. Collect the aqueous fraction. Add 200 μl chloroform. Pulse vortex for 3–5 sec. Centrifuge 8,000 × g for 5 min at RT.

e. Collect the aqueous fraction. This is final Virus Nucleic Acid.

The final viral nucleic acid goes through the Qiagen DNeasy Blood and Tissue kit.

I am not sure if the final nucleic acid contains RNA. If it does not contain RNA, I would like to know at which step I should put my sample through the kit.

I read protocols using CTAB to extract RNA as well as DNA, but I am not so sure about phenol:chloroform:isoamyl alcohol or plain chloroform. I read a similar protocol for extracting RNA that used CTAB and phenol:chloroform:isoamyl alcohol, but they replaced the chloroform with isopropanol and centrifuged, collecting the pellet as final RNA.

If someone could help me sort this out, it would be great!

FYI, this is part of a protocol to enrich for viral particles and extract the nucleic acid from stool with the least amount of human or bacterial contamination.

I have performed RNA extraction of blood sample using TRIzol method. I ran RNA samples on non-denaturing gel (1.5%) that showed extreme DNA contamination and degraded RNA band. Please suggest me how should I overcome this issue.

I have used STAT60 to isolate RNA successfully. I am wanting to collect RNA and protein from tissues, and according to the manufacturer's information I could allegedly use STAT60 to collect both types of molecules. What I would ideally like to do is precipitate both RNA and protein from the same tissues and submit the protein for mass spectrometry. Has anyone used STAT60-isolated proteins successfully for mass spectrometry?

I tried to use RNAase to study protein and I have tried to digest protein to get nucleic acid also. The problem is yield is low and RNA is fragmented. I am trying to purify both of them in functional form. Can anyone please guide me with it? Thanks a lot.

I am currently collecting 1k-100k cells through FACS sorting for qPCR and RNA-seq. Several online protocols suggest sorting cells directly into TRIzol, and I have found this method effective for cell collection and RNA extraction. However, I am concerned about the aerosolization of TRIzol during this process. Does anyone have suggestions or best practices to prevent TRIzol aerosolization during FACS sorting?

Hi,

I extracted total cellular RNA from HEK293T cells by using a commercial kit. I left samples untreated or treated with 2ul RNaseA/T1 mixture (invitrogen) in the presence of PBS or protein lysis buffer NP40 (because I will use RNase in the presence of lysis buffer later). The labels of shared gel pic are as follows:

1) 1kb NEB DNA ladder

2) 500ng total RNA

3) 500ng total RNA incubated at 37C for 30min in the presence of water (untreated)

4) 500ng total RNA incubated at 37C for 30min in the presence of 1X PBS (treated)

5) 500ng total RNA incubated at 37C for 30min in the presence of lysis buffer (treated)

6) 100bp Promega Benchtop DNA ladder

1) Why there is only one thick band in 2 and 3 but no band or smear in 4? Why there is a band shift in 5?

I ran non-denaturing 1% agarose gel in TAE buffer at 30V.

I want to check mRNA expression levels in these NPWT samples. Is there any way to isolate RNA from these? I tried TriZol based extraction but all I got was a smear (considering the samples were thawed multiple times, I expected this).

Thank You

I am having problems with the stability of a microRNA against exo/endonucleases in serum. I have seen that there are several modifications to make it more resistant, including modification with phosphothiorates. My question is where and how to place these modifications along the RNA strand and how can they also affect the recognition by intracellular RNase H?

Thank you!

I have performed 3 RNA isolations on Jurkat cells using the phenol-chloroform principle, using Omega Bio-Tek RNA Solv® & following the protocol. I took care in not disturbing the separated phases when removing two thirds of the aqeous phases but the data I get when I measure the 260/280 & 260'/230nm absorbance ratios lead me to suspect contamination of the samples with phenol or other reagents.

Most of the samples have a 260/280 ratio below 1,6 and only one sample has a 260/230 ratio that comes close to 2 (1,84 to be exact). Subsequent cDNA synthesis & RT-qPCR have not resulted in genes of interest to show any fluoresence at all, only the housekeeping genes in 2 of 7 samples showed up. Problems with primers & RT-qPCR were ruled out as the same setup yielded viable results previously.

Thanks in advance!