Questions related to RNA Isolation
Revered researchers , I intend to isolate RNA from oral squamous cell carcinoma surgically resected sample. What is the best and economic kit I can use for RNA isolation.
I found the following:
RNAzol® RT - 50ml = 9.6 INR
GenElute Total mammalian RNA purification kit (70purifications)= 40k INR
RNA isolation kit From Sigma Aldrich = 40k INR.
Please suggest me the one I should purchase. I’m a novice in this.
I'm fractionating cells and pulling down a viral protein that binds to a specific RNA. I've been trying to extract the RNA but I have no luck. I have tried basic trizol extraction and direct-zol RNA mini prep with no luck. I think the issue comes when I turbo DNAse treat my sample after RNA extraction. I even clean up the sample and there is no RT-PCR product in my control. What should I do? I was thinking of phase separating the trizol with chloroform first and then adding the ethanol and incubating overnight in -20C before doing the zymo kit. What do you think?
I would like to know if you are using RNase to digest RNA outside EVs? If yes, how do you stop RNase before exosomes lysis to protect RNA inside?
I accidently added five fold more chloroform in the initial steps of RNA isolation. What are the consequences of this? Also, if i add more isopropanol to compensate this effect, how both chloroform and isopropanol are related ?
I usually isolate both DNA and RNA with Qiagen AllPrep kits without problem when frozen samples in RLT buffer.
However, I have a sample frozen in RLT buffer without previous removal of cell culture media. Is there any option to rescue the sample?
Qiagen's protocol said: "Incomplete removal of the supernatant will inhibit lysis and dilute the lysate, affecting the conditions for binding of RNA to the Rneasy silica-gel membrane. Both effects may reduce RNA yield."
I have tried to wash the sample with PBS after thawed, then resuspend in RLT and continue with the protocol, but it didn't work.. How can I isolate DNA of this sample?
The agarose gel run of RNA samples before DNase treatment gives proper bands for RNA but after DNase treatment, the RNA samples on the gel run band are coming below the ladder size ( I used a 1Kb ladder). I treated RNA samples with DNase at different concentrations of 10 units, 20 units, 8 units of enzyme, and at different incubation time also 30 min. and 25 min. at 37 degrees followed by RNA isolation by Trizol. Yet I am getting the degraded RNA. I am open for your valuable suggestions.
I've been working on improving RNA quality lately since I've had a number of bad qPCR results in the past few months. We use the Trizol method in our lab, and recently there has been a lot of precipitation during the RNA isolation. Brief rundown, we homogenize our tissues (I'm using mouse hearts and fats) in Trizol per manufacturer's instructions, followed by a spin to remove insoluble material, and then an addition of chloroform to isolate the RNA. After spinning and removing the aqueous phase, we add it to isopropanol. At this stage, my samples turn extremely cloudy, obviously containing some kind of precipitation. This precipitate can be spun down, and initially forms a gel-like pellet which turns opaque white after washing with 70% EtOH. There is not supposed to be cloudiness at this stage though...the solution is supposed to turn clear after adding the aqueous phase.
I tried this with a blank set of samples (i.e. Trizol without homogenized tissue) and I got the same result...the aqueous phase I pulled off of the blank becomes cloudy after adding to iso, and can be spun down to produce a pellet that looks just like what came out of my tissue samples. I should also note that in my tissue samples, I could see a tiny blue dot after the first EtOH wash (we use glyco blue to precipitate with RNA), so there was definitely a good RNA pellet in there, but it was surrounded by a big flaky piece of white precipitate. The A260/230 ratio is also very low with RNA I get from these cloudy samples, usually around 0.1 (260/280 is good at around 2.0).
Has anyone else had this issue before? Is this some kind of salt precipitation or something else, and how should I get rid of it? I'm guessing maybe the Trizol got contaminated since it happened with blank samples (and I used fresh chloroform and iso, and adding chloroform straight to iso without Trizol did not form this cloudy precipitation), but before I throw the bottle out I'd like to see if this is a known issue I can fix. Any advice is appreciated!
It's about MagMAX™ mirVana™ Total RNA Isolation Kit. Due to lack of samples, I want to use tubes instead of plates for manual extraction. Protocol suggests, a plate shaker at 700, 950rpm etc etc. Is there any alternative equipment I can use?
One of the people in my lab added too much isopropanol to an RNA sample that was isolated with chloroform. What can I add to reduce the percent isopropanol? What is safe to add to an RNA sample/precipitation reaction? The sample was isolated with chloroform, so can I add chloroform to reduce the concentration of isopropanol?
Our lab has some leftover TRI solution and column-based kit for RNA isolation. Is it possible to use them together? Could i use the TRI solution to homogenize the sample then followed by column purification from the column-based kit?
We work with plant samples with some limited access to refrigerated centrifuge. With reduced centrifugation time i could use the regular centrifuge instead.
As per the kit protocol for the longer durability use RNase-free H2O (with 0.1 mM EDTA) or TE buffer (10 mM Tris, 1mM EDTA) to preserve the RNA for a longer time, but I have a doubt about how to restore the RNA from the above soln?
Kindly suggest the standardized or best protocol.
We are digesting mouse cortices and trying to extract the RNA from microglia for sequencing. We have gotten the isolation working to get good yield of cells (about 200,000 for both cortices) but the RNA after flow sorting is having really low RNA integrity scores. We are thinking that this is because of the sort process and are thinking about using RNALater for preservation of cells prior to sorting (i.e sorting with RNAlater as the buffer!). Has anyone tried this? If so - does the RNALater degrade the fluorophore signals? Any other tips for isolating RNA from sorted primary cells would be appreciated!
I was wondering if anyone knows a way I could Isolate RNA and Protein from the same cell pellet. I'm not looking for a kit that allows me to do both at the same time unless that is my only option. Basically I have cell pellets I have stored at -80 degrees that range from 800,000-9x10^6 cells per pellet. Because I really only need about 100,000 cells for RNA isolation for qPCR I dont want to waste an entire pellet of cells on just RNA. I need several million cells to achieve enough protein for my western blot analysis. So is there a way I could resuspend this pellet and potentially use like 20% of the lysate/resuspension for RNA and the other 80% for western blot analysis at a later time without damaging the cells or compromising one procedure for another? My big concern is the lysing of the pellet with one type of buffer/breaking the cells and it compromise either of the procedures...
I am trying to isolate RNA from monocytes, but using the Macherey-Nagel Kit (NucleoSpin RNA Plus) I only seem to get concentrations of 10-20 ng/µL RNA in 20 µL elution volume, which is barely sufficient for my follow-up experiments.
I culture 750,000 monocytes in 500 µL RPMI 1640 medium, supplemented with GlutaMAX, NaP, gentamicin and human serum for 24 hours at 37 oC. Then I harvest them by adding LBP lysis buffer to the cells (and resuspending thoroughly) -> freezing them in liquid nitrogen and then storing at -80 oC until subjected to RNA isolation.
How can I get a higher RNA concentration?
I have been trying to isolate total RNA from cattle whole saliva samples for gene expression studies. Saliva samples were collected early morning before feeding to the animal and collected in Trizol for Trizol isolation method and in RNA later for kit method. I have tried both the Trizol isolation method as well as the kit method (Qiagen RNeasy Mini Kit) to isolate RNA from the saliva sample (Pellet as well as Cell-free Saliva) by applying all precautionary measures. After isolation when I run RNA on agarose gel (1%)18s and 28s bands are not visible but I'm getting a band ~100bp. Nanodrop reading OD (260/280) I'm getting around 1.7 to 1.9 with concentration varying from 110-200 ng/microliter. After cDNA preparation, I'm not getting any amplification for GAPDH and ACTB genes. Can anyone suggest where I'm making mistake and how I could enhance my RNA yield?
In the attached gel image Last 3 lanes consist of isolated RNA and the other lanes consist of PCR amplification result of GAPDH and ACTB in which no amplification was observed.
Consistently I'm getting this result for RNA Isolation. Please help.
my aim was to measure miRNA expression in exosomes. I used TRIzol method for RNA isolation and miRC_LNA_miRNA RT and PCR kit for reverse transcription and qPCR as follows.. however, my qPCR was not successful. most of the tested samples including positive control (U6) were giving me either weird Cq values like 7 to 14ish or not determined at all. Does it mean I did not have enough cDNA generated or what else can be considered to look at?
has anyone had this experience or any recommendations would be great. thanks!
I have extracted DNA from my soil samples (dry and sandy) through two different extraction kit (both from Qiagen)
1) by DNeasy PowerSoil Kit
2) by Total RNA Isolation kit (DNA eluting after RNA isolation through eluting buffer).
I got huge difference in extracted DNA concentration. Total RNA Isolation kit did not have good amount of DNA, but same soil sample had really good DNA concentration when extracted by PowerSoil kit.
Does anyone know what is problem here and which result can be more reliable?
During my qPCR, I run no-RT (NRT) samples and no-Template Control (NTC) samples along with my normal cDNA samples containing animal tissue. However, I got amplification values for all my NRT samples, but none for my NTC control samples, which probably indicates genomic DNA contamination in my RNA samples.
However, the Macherey-Nagel kit which I'm using for RNA isolation contains gDNA removal columns which should normally remove any genomic contamination. Assuming the handling of my samples went fine, what else could have contributed to those problematic results?
I would also be interested to hear any ideas on how to remove gDNA from the rest of my RNA samples - after using the kit and gDNA removal columns - with little to no risk of RNA degradation.
Thank you in advance!
I am trying to isolate RNA from freshly isolated neutrophils from adult blood. Further I maintain culture for 3 hours hours according to my need. I use trizol choloroform method. I got very poor yield, A260/280 is also less than 1.8 even. No result was obtained on agarose gel. Can anyone please share your protocol or suggest me a good idea what will i do. Thanks in advance
Hello researchers. I am using CTAB method to extract total genomic RNA from Citrus leaves. I often observe that the pellet is very minute and keeps floating which makes it difficult to discard the supernatant. I want to know what could be the possible reasons for getting a floating pellet and how to avoid it. Thank you.
After adding .5 μL isopropanol in my sample i see 2 blurry clouds (one in the bottom and the other one in the top) in my sample. Anyone knows why this happens?
I would like to know what the reason of the shape of this curve in the spectrometer after total RNA isolation using Trizol?
I would like to know what the reason of the shape of this curve in the spectrometer after total RNA isolation using Trizol.?
So the whole blood samples are in RNA later solution and kept at -20 (500 ul blood+ 1000 ul RNA later). Please suggest protocol to discard the RNA later and process for RNA isolation. These RNA samples will be used for both sequencing as well as for cDNA synthesis
N.B: RNA later solution is from invirogen and the RNA isolation kit I will be using is from Qiagen.
Any help would be appreciated
When performing Viral RNA extractions I perform a negative extraction using water. This is then run as a negative control for my RT-PCR reaction. Recently this control has been showing up RNAse P positive. In addition, when I run the same extract negative sample in duplicate on my PCR plate, sometimes I get RNase P in only one of the duplicates. What is happening? It only ever happens in the Extraction Negative, it never occurs in my PCR negative control.
This issue is occurring for multiple technicians.
Which of the two kits is better for RNA isolation from plant rich in secondary metabolites? Maybe you could recommend me something else? I would like to obtain high quality and quantity RNA for qRT-PCR. My material are leaves, stems and roots of Salvia miltiorrhiza.
Hi, I have been doing RNA isolation using the standard RNAeasy Plus mini kit. The 260/280 ratios seem fine - between 1.8 - 2-0, however the 260/230 ratios have been consistently low ( below 1.. as low as 0.3) or extremely high( above 2 or even 3) and I am unable to figure out why. I was using the same kit in my previous lab and following the protocol but I never had this issue. I do the extra spin step with an empty eppendorf to make sure that I have dried the column before elution with water.. I tried running a few of the samples on the Bioanalyzer to check the RIN values but the samples look fine..
I asked around in the department but either people were not facing this problem or they told me it did not matter so much .. so I am confused.. AND curious .. as to why this keeps happening with my samples.. what could i be doing wrong.. ? when I measure the RNA amounts on the nanodrop, I always keep the samples on ice and then I store them at -80deg C.
Would appreciate your suggestions / thoughts.
Hi, I want to ask that QIAGEN ask following steps to do but if we dont have needle & syringe so can we directly put RNase free water into DNase I stock ?? If not so what is alternative of this? ''Prepare DNase I stock solution before using the RNase-Free DNase Set for the first time. Dissolve the lyophilized DNase I (1500 Kunitz units) in 550 μl of the RNasefree water provided. To avoid loss of DNase I, do not open the vial. Inject RNasefree water into the vial using an RNase-free needle and syringe. Mix gently by inverting the vial. Do not vortex.
My 2nd question is that in this step we keep it at Room temperature for 15 min? or centrifuge it at 20-30 degree for 15 min?
Add the DNase I incubation mix (80 μl) directly to the RNeasy spin column membrane, and place on the benchtop (20–30°C) for 15 min.
Today I was isolating RNA from human atherosclerotic plaque tissue. During the process, I found the interphase extremely thick. Does anyone know of a reason what could cause this? Does it have further effects on the RNA isolation?
Hi everyone, I am dealing with C2C12 lineage, and I am standardizing the number of wells needed for PCR analyses. I tried to isolate the RNA with trizol protocol for cells, although until now I didn't find any articles that explicit show the exact RNA yield I will need to found at the last step of quantification. I only found it for fibroblasts, epithelial cells. I wish someone can help me about it.
Best wishes for everyone.
I am working on a 3D hydrogel system. I plan to study changes in gene expression in my 3D hydrogel sample. My question is, can I isolate cells from the hydrogel, then culture for 6hrs and then initiate my RNA isolation and downstream assays? What are other possible methods to isolate good quality RNA?
Thanks in Advance!
I have been using the Qiagen's RNeasy kit for RNA isolation from mealybug "Maconellicoccus hirsutus". Though the 260/280 ratio always lies between 1.8-2.0, the 260/230 values are always less than 1 and sometimes even between 0.2-0.5.
Is it because of wax coating on the adult females?
I'm trying to improve some protocols for RNA isolation from (among others) adipose tissue . I'm trying to figure out what are the best binding conditions for RNA using qiagen silica columns.
I did the extraction of RNA using combined protocol from different publications and qiagen kits which use trizol(qiazol etc) + chloroform + columns. Results were not good, especially the 260/230 proportion is very low eg. 0,3 so it cannot be used in qPCRs.
Also I tried two step method for RNA isolation - first homogenisation with buffer cont. Gu-HCl, Tris-HCl and triton x-100 pH 6,6 then after centrifugation supernatant was applied to qiagen column to theoretically bind DNA (?), then EtOH was added to the flow-through to precipitate RNA (is it better to precipitate it through the night at -20?) and washed with buffers from Roche or Qiagen. Of couse I know that they might appear DNA contamination but it will be removed with dnase treatment. RIN are very good - 8,2 or even higher usually nothing less than 7, but the concentration is very low (20ng/ul) and 260/230 too.
So I'm looking for some advices, books, articles, websites - everything :) to improve my work. Or maybe someone have any ideas or can explain me some things?
What's yours expierience with trizol-chloroform-columns extraction? Did you do the two-step RNA isolation on columns?
I have about 100 blood samples in edta tubes, I keep these tubes at -80 degrees. I'm going to do gene expression from them,
Has anyone used GeneJET Whole Blood RNAPurification Mini Kit
#K0761 and had good results? Or should I do RNA isolation with trizol?
I would like to isolate total RNA from single cells for qPCR (or even sequencing later on). What protocol or kit should I use to obtain good quality RNA with minimum loss?? I've seen there is a kit "PicoPure® RNA Isolation Kit" but I'd like to know if there are other suggestions before buying such and expensive kit..
Can I store mouse ear tissue homogenate at -80 before RNA isolation using Gene JET RNa purification kit(cat no# K0731)?
Using H9 hESCs I have developed neural progenitor cells using SMAD signalling inhibitors, then differentiated them into neurons by excluding EGF and FGF from the media and plating onto laminin coated plates. I have very nice neural networks in the dishes but when I go to isolate RNA using Qiagen RNA easy mini kit, I dont get any RNA. I was wondering if anyone else has had this problem with RNA isolation from neurons and if there's anything I can do to rectify? Thanks in advance for any advice!
I'm planning to isolate RNA from cells stored in RNAlater with Qiagen mini kit. In one article, I saw we can directly use cells in RNAlater without washing it with PBS (but needs to put a higher volume of lysis buffer). Does anyone has done that?
We have a RNA collection in our freezer and wondering if it’s worthwhile keeping this study or throwing it away to create space for other studies.
The RNA was isolated 20 years ago from heparin blood tubes. The time until RNA isolation differs between samples from 2 to 5 hours. The RNA has been stored at -80C.
We know it’s best to use Paxgene tubes to stabilize the RNA. Would any of you still use these RNA samples for transcriptome studies or any other types of studies?
Does the heparin have an influence on the outcome in any way?
Many thanks in advance.
I want to delve deeper into nanopore sequencing and bacterial transcriptome analysis and as I don't have that much experience in this area I am basically venturing blindly.
I isolated my RNA from E. cloacae using column Zymo kit. Then I tried to measure my results in the Tapestation, however I obtained suprisingly low RIN scores (RIN: 4-7). I recheck my samples for DNA contamination and RNase with Qubit and my samples are fine. Obviously I have spikes where I should not have them but what would possibly cause my RNA to be so damaged?
Is anybody has any idea what could be the issue? I am attaching my Bioanalyzer results from 2 bad samples and one that is slightly better.
Thanks for any help in advance!
I am doing a dissection to get a small amount of tissue for RNA isolation. The saline was made using non RNase-free/sterile parts. Also, the area I am dissecting is very close to the gut which occasionally becomes permeated so there is a likelihood of high levels of RNases during the dissection.
Somebody suggested I add BME to the saline to inhibit RNases. My question is what would be a sufficient amount to add to inhibit RNases without making it immediately toxic to the tissue I'm dissecting.
I am looking for synthesizing cDNA from total RNA, isolated from wheat leaf. I have GoScipt Reverse Transcription Kit. After some searching, I have found that this MMLV reverse transcriptase does not have RNaseH activity nor the kit has any separate RNaseH enzyme.
Can I use this kit for synthesizing full length cDNA for cloning purposes? Or I have to use separate kit/ RNaseH?
N.B. My target gene is 1.6Kb in length
Hi, for my experiment I have to seed the cells on the lower (abluminal) side of the inserts (no cells on regular side). I am facing issues with isolating RNA from those cells. Anyone has any experience? There should be around 300,000 cells (on 0.4 um Falcon 12-well format inserts). I have tried Qiagen RNeasy columns for RNA isolation. Thanks.
Hi I'm isolating RNA from mycobacteria for qPCR
so I added 500ul of Trizol to cells and got rid of cell debris with centrifuge
after that I added 300ul of chloroform and centrifuged 5min at 16000g, 4℃
but i realized that aqueous phase was so cloudy and the interphase and aqueous phase wasn't separated properly
so it was really hard to get aqueous phase without cloudy things
should I centrifuge longer than that?
is there anyone who experienced similar problem?
I wanted to start this discussion to gather the experiment results from whoever have gone through this.
I am trying to extract total RNA from both serum and plasma, continue with cDNA synthesis and qPCR.
I am using grade II or III cancer patients’ blood sample.
I am on my validation period and still working on it.
Could you please share your experiments in this field ?
All you experiments such as:
- Serum and Plasma isolation protocol,
- Amount of serum and plasma used in RNA isolation,
- RNA extraction kit? Did you use specific kit for RNA isolation from serum/plasma or any other commercially available ones?
- Any key point in cDNA synthesis,
- Probe or syber green, which one did you chose for qPCR experiment?
- Any commercially available qPCR master mix or more specified one?
Hi there, I'm looking for help to deposit RNA from DEPC-treated water.
I used Trizol to do the total RNA isolation and finally dissolve the RNA in DEPC-treated water. However, I found I did a wrong calculation about the volume of the DEPC-treated water, which cause the low concentration of RNA. How can I make the RNA be precipitated from the DEPC-treated water? It would be pretty kind if someone can help me out.
I am trying to extract RNA from Aedes aegytpi to analyze gene expression in genes associated with the detoxification of insecticides. I am using the SV Total RNA Isolation System from Promega, but I got a very low yield of RNA (from 30 - 50 ng/ul). Can you share any tips or protocols or how have you solve low yield in RNA isolation from mosquitoes. Thank you very much.
I am trying to run a qPCR on RNA isolated from FACS samples but it seems that the quality of my RNA / cDNA is not high enough and the qPCRs keep failing. My primers are not the issue since they work on RNA isolated from tissue.
I isolate the RNA thanks to the QIAGEN RNeasy Micro Kit and already tried to improve the quality of the isolation by:
- Combining the RNeasy Micro Kit with a Trizol RNA extraction
- Directly collecting the cells in Trizol to avoid its dilution in buffer
- Extra PBS washing steps before proceeding with the RNA isolation
Unfortunately, this didn't improve the qPCR results.
Would anyone have had the same issue and could find a solution?
Hello everyone! One of my students asked me a question about DNAse step during RNA isolation. She asked me how is it possible, that DNAse is responsible for hydrolyzing phosphodiester bonds in DNA, and it does not degrade the same bonds in RNA, although it is a less stable molecule? I haven't found any precise answer, so I would be very grateful for your help!
My problem is on the purity of my RNA. When tested on nanodrop, the A260/280 ratio is around 1.8 - 2.0 which is good; however, the 260/230 ratio is low, around 0.4-0.5. Any tips on how I can purify my RNA? FYI? I'm using Trizol reagent.
I guess there should be a washing/equilibrating step (buffer) and an elution step (buffer). I have tried direct elution with warm TE with limited success.
1. We have extracted DNA from nasal swabs and BAL using the Qiagen DNA Blood and Tissue Kit for extraction. There is now a Qiagen QIAamp DNA Microbiome Kit available. Has anyone compared DNA yields with the DNA microbiome kit versus the Blood and Tissue kit?
I have been having great difficulty in isolating RNA from bovine preantral follicles despite many changes to protocol. Our goal is to isolate RNA from a single follicle with ~100 cells, but even with 10+ follicles pooled, our RT-qPCR results have been negative for both beta-actin and aromatase expression but is able to detect expression in a single oocyte with a few granulosa cells attached. We believe there is a lysing issue with the follicles due to the basement membrane. I have tried: 2 different single-cell RNA isolation kits, using collagenase/pronase for 30 mins before lysis step in RNA isolation, adding more cDNA, increasing to 40 cycles, low retention tubes, using Cells-to-cDNA kit, and using RNA-to-PCR one step kit. Any advice for getting this to work is greatly appreciated!
I am a Master's student working with Murine Skin samples for qPCR analysis. I am having a hard time getting clean ribosomal bands with my RNA- some appear but are still VERY smeared. I have good A260/280 ratios (around 2). I am homogenizing using a drill and lysis buffer + Proteinase K for 30-45 minutes. I then pull off the supernatant and isolate my RNA.
I have attached an example of what my bands look like. I seem to be getting good amplification from the cDNA made from the RNA, but I would like to clean it up before I publish with it.
Thank you in advance!
For several months, we have been collecting whole blood from patients then using density gradient centrifugation to isolate mononuclear cells (MNCs). We then immediately add Qiagen's RNALater to the cells and store them at -80. We have started trying to isolate genomic DNA and RNA, the first for HPLC and the second for microarray. However, there are red blood cells and (probably) hemoglobin still present that clogs up Qiagen's AllPrep DNA/RNA Mini Kit columns. We tried starting with the first few steps of the Trizol method to help clean the samples prior to launching into the kit, but were wondering if there is a better way. When speaking with a Qiagen rep, my colleague was told that the their red blood cell lysis buffer was not compatible with the RNALater we have been using.
I will be performing RNA isolation with frozen lung biopsy samples and am in the market for a tissue homogenizer. I am wanting a homogenizer that will fit in a 1.5ml tube. I was looking at the Fisher 150 Homogenizer (15-340-167), as well the 7mm Fisher saw-tooth probe (15-340-174). If anyone has experience using these products or knows of another product that will work, please let me know.
Hi, I have been struggling with a weird issue for my RNA samples. I used the Qiagen RNA plus mini kit. For some reason no matter how careful I am with the washes, when I measure the RNA, I get really low values for the 260/230 ratio, on average around 0.5 or below. I follow all the steps suggested in the kit.. I thought that it must be the Ethanol which causes such ratios so I try to adequately dry the column with an extra spin.. but that does not seem to help.
I checked the quality of my RNA on the Bioanalyzer and it seems the quality is fine. Even the yield of RNA from my samples is decent .. ( I grow cells in 6 well plates ).
I have limited amount of samples, so I am trying to get RNA, DNA and protein from the same samples. I used Trizol reagent for RNA isolation, then use back extraction buffer for genomic DNA isolation. After back extraction I saved the viscous pellet, I have seen people save it for protein isolation but was not able to find a protocol to proceed. Can anyone help me with this?
Currently, I have a problem to isolate total RNA from mouse primary cortical neuron culture. I am seeding neurons to poly-L-lysine coated coverslips and seeding 500k cells per coverslips in a 6-well plate. For RNA isolation, I was scaping 3 coverslips under 1 ml trizol. After that, I was proceeding with Direct-zol RNA isolation kit protocol. But there was no RNA or sometimes very little RNAs such as 20ng/ul after isolations at many times. Cells were looking confluent and healthy before scraping. Therefore, I am not sure about which stage the problem occurs. I would be so grateful to get an extra idea/tip about this. Is there anyone doing RNA isolations from primary neuron culture and managed to solve this kind of problem?
My university is starting to apply a new phenol-free policy and I am trying to find a phenol free RNA-isolation kit. It would be for brain tissue. Normally I use Trizol/Qiazol reagents getting a good yield. If anybody is using one that it's working well for brain, please share :)
Many thanks in advance!
We're doing a RNA phenol/chloroform extraction from individual zebrafish embryos and using a phase lock gel to get the best possible RNA quality. We'd like to also be able to genotype the embryos by DNA extraction after this. I've seen protocols that allow back extraction of DNA after RNA isolation, but they don't use a phase lock gel. Anyone have any idea if this can be done still using the gel?
I did a few rounds of RNA isolation today and found something very strange. I usually have very good A260/A230 ratios along with A260/A280 ratios, but this time around my A260/A230 ratios were VERY low. I am not sure what went wrong but it was consistent among the two times I isolated today.
Could this be due to the reagents in my RNA isolation kit being contaminated? This doesn't seem likely to me but this is really the only variable that has changed considering I have moved on to use the newer kit now that I am proficient at isolation.
In my second run today I tried eluting two rounds with water at the end which helped a little but the ratio was still far too low. It also dropped my concentration down to about 30 ng/ul from 100 ng/ul.
In past isolations, my concentrations would range from 400-1000 ng/ul, so this was very strange.
Tomorrow I plan on trying it again with a different kit to see if that was the issue.
Any suggestions or comments would be appreciated, thank you!
I am trying to determine what best time-point to harvest my cells, HMVEC, after transduction with long non-coding RNA to determine changes in mRNA levels, specifically VEGFR2/KDR.
I am capable of achieving ~90% transduction efficiency by assessing GFP+ cells via fluorescent microscopy as my adenovirus is tagged with GFP. My current protocol is:
Day 0: Seed cells
Day 1: transduce cells with adenovirus (24 hours post seeding)
Day 2: change media (24 hours post transduction)
Day 3: harvest cells
What would be an ideal time point to harvest cells to determine changes in mRNA levels? I was thinking between 16-24 hours post transduction. Would appreciate any input or advice, thanks in advance!
I am about to IP my protein of interest and maybe (I hope so) some small RNA would co-IP with it. Since this is my first time in this area, would anyone be so kind to suggest the best method for separating these RNAs for the further analysis (based on your own experience [good & bad])? I am thinking of trizol but not really sure.
Thank you in advance!
I extracted total RNA from fungal samples and my goal is to send the RNA for RNAseq, however my RNA did not pass the quality control due to atypical baseline by the bioanalyser results (Please see image attached).
The total RNA was extracted using the QIAGEN RNeasy mini kit and DNase treatment was done after RNA extraction using the PROMEGA RQ1 RNase-free DNase.
Does anybody know why I am getting this atypical RNA quality results? Thank you very much!
I've been trying to isolate total RNA from guinea pig's knee cartilage. I've tried Qiagen RNeasy kit, miRNeasy kit, trizol method with lots of modifications and have also used the RNaqueous total RNA isolation kit from Thermo Scientific but I've not been able to achieve RNA ratio of more than 1.4 for cartilage and 1.6 for synovium. In addition to the integrity the RNA conc. is also very low because the amount of cartilage available from the knee of guinea pig is very less (<15mg).
I homogenise the samples using Bead beater (bead mill).
I am doing silica magnetic beads-based RNA isolation. I am facing some impurity issues because of washing. So kindly share some best washing buffer composition of wash buffer for especially for RNA isolation.