RNA Isolation - Science method

Hi,

I'm currently working on the analysis of wastewater treatment efficiency on viruses. I have spiked my wastewater (abbatoir water from fish slaughterhouses) with BCoV and TV prior to treatment with standard chemical coagulants containing iron and aluminum, and will now evaluate the efficiency by RT-PCR.

The issue is that my RT-PCR results are fluctuating and are often resulting in No Cq, especially on the treatments where max amounts of iron are used. I think the problem might be innhibitors from the coagulants. I'm currently using QiAmp viral-RNA isolation kit and RNeasy clean-up kit.

Has anyone working with samples containing chemical treated wastewated experienced the same issues? If yes, do you have any recommendations to other kits/procedures I can use to clean my samles sufficiently to isolate the RNA?

Thank you in advance.

Best,

Mia Tiller Mjøs

Hello,

I performed an rt-PCR using CRISPR KO cells as I have done numerous times before. I am confident that there has been no error in RNA isolation, cDNA synthesis protocol, or primer usage. However, I did use a brand new cDNA synthesis kit during the cDNA synthesis step. Could this be the cause for my CT values being too high? I cannot think of another reason as I have used these primers with this CRISPR cell line in the past with no issues. Any help will be greatly appreciated!

Does cell lysis affect RNA isolation? I ve done my cell lysis by vortexing after that i ve done RT but im not geting the results.

Hi,

I'm trying to isolate RNA from cell cultures using the Trizol method. However, all my samples have low 260/280 values (~1.6). Could this be because I used chloroform and isopropanol, which was not molecular biology grade? For RNA isolation, I used samples that were kept in Trizol at -20C for about 2 months.

I have nhe6 KO mice, confirmed via genotyping from their tail clips. I isolated BAT, iWAT, and gWAT from three controls and three KO mice. RNA isolation followed by cDNA preparation was performed from these isolated tissues. On running qPCR, I expected low ct values (high nhe6 expression) in controls and high ct values or undetermined ct values (may be?) for KO samples. However, shockingly! I got similar ct values (30-32) for both- controls and KO.

The questions here is-

Is it correct to perform qPCR on genomic knockouts? AND WHY?

I am trying to Isolate RNA from sugarcane. The objective is to study the involvement of genes in red rot diseases.

Plant stems are inoculated with Red Rot fungus inoculum. Plants are mature and it's been 2 years of their growth.

The protocol I follow is as

1.                  Add 0.5 ml of cold (4ºC) Plant RNA Reagent (TRIzol from Invitrogen) for up to 0.1 gm of frozen, ground tissue. Resuspend the sample thoroughly by briefly vortexing or flicking the bottom of the tube.

2.                  Incubate the tube for 5 min at room temperature. Lay the tube down horizontally to maximize surface area during RNA extraction.

3.                  Centrifuge for 2 min at 12,000 x g, transfer the supernatant to an RNase- free tube.

4.                  Add 0.1 ml of 5 M NaCl to the clarified extract and tap the tube to mix.

5.                  Add 0.3 ml of chloroform. Mix thoroughly by inversion.

6.                  Centrifuge the sample at 4ºC for 10 min at 12,000 x g to separate phases. Transfer the top, aqueous phase to an RNase-free tube.

7.                  Add to the aqueous phase an equal volume of isopropyl alcohol. Mix and let stand at room temperature for 10 minutes.

8.                  Centrifuge the sample at 4 ºC for 10 min at 12,000 x g.

9.                   Decant the supernatant, taking care not to lose the pellet, and add 1 ml of 75% ethanol to the pellet.   Note: The pellet may be difficult to see.

10.              Centrifuge at room temperature for 1 min at 12,000 x g. Decant liquid carefully, taking care not to lose the pellet. Briefly centrifuge to collect the residual liquid and remove it with a pipette.

Add 10-30µl RNase–free water to dissolve the RNA. Pipette the water up and down over the pellet to dissolve RNA.  Store at -70 ºC.

Before starting the protocol we need pestle, mortars, tips both 1ml and 200ul, Eppendorf, and PCR tubes washed with DEPC-treated water. DEPC 1ml added to 1-liter water makes the DEPC treated water.

5ul of loading dye and 5ul of RNA sample was run on 1% Agarose gel in TAE buffer for 50 min at 60 voltage, 1kb ladder was used and I did not use the Denaturation method for gel.

Attached are the pictures and I am not getting any results kindly help me with how to proceed.

I am going to sacriface my animals in a couple of weeks and dissect plenty of tissues in a day. There is no way to do all protein and RNA isolations at the same day. Therefore I must freeze (-80C) some tissues for at least a few weeks and isolate part by part.

Could you please enlighten me about RNA-later solution (invitrogen-thermofisher)? Can I put my tissues into RNA-later solution directly or required to homogenise my tissues in RNA-later solution before freeze it?

Also I am quite concern about protein extraction from tissues. Can I extract proteins from frozen tissues and if it is possible what kind of solution require to freeze tissues? Or freeze it without adding any solutions?

While lysing the leaf sample for RNA isolation a jelly-like substance was released which led to a low yield of RNA. How to overcome this issue.

PS: RNA isolation was carried out using nucleospin RNA kit

Hi!

I have samples with callus remainings and bacteria that have been cultured on a callus solution. Now I want to isolate only the RNA from the bacteria to eventually only get mRNA from the bacteria to be able to RNA-seq. Now the problem is that the remainig callus appears to contaminate/overrule the RNA-seq data for now. So therefore, we would like to isolate the bacterial mRNA and remove all the mammalian parts.

If anyone knows something, let me know! :)

Thanks in advance!

I was isolating the exosomes from 40 mL of urine sample by ultracentrifugation method and further try to isolate total RNA from it for NGS. I was able to isolate total RNA conc. around approximately 5-9 ng/uL using mirVana kit. I followed the all precautions during isolation such as using ice all the time, and RNaseZap (decontamination solution). I also went for the RNA QC in which RIN NO was 1.5 due to which NGS was not possible. Please suggest me how will I improve my urinary exosomal RNA concentration or any alternative technique to do urinary exosomal microRNA profiling.

So, i would like to know what concentration of Tris HCl i have to prepare in order to use it in RNA isolation protocol? Additionally, i would like to know why to use this buffer, is it only for pH stabilization?

Thank you,

Kiriakos Athanasiadis

I have performed RNA isolation from brain tissue using trizol and a commercial kit (columns). I got good RNA concentrations (>600ng/uL) and 280/260 ratios (near 2,0) in all samples. However, 260/230 ratios were abnormally high (3-9).

What is the cause of these values? How can I improve the protocol?

Thanks for your help!

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.

Thanks,

Zankruti

I used QIAGEN RNeasy Mini plus Kit for RNA isolation. The final step require me to elute RNA into 1.5 ml centrifuge tube provided by this kit.

I am wondering:

1. Can I store the centrifuge tube under -80 °C for long time storage? (in the protocol, it is said -70 °C though)

2. what is the difference between cryo vial and centrifuge tube?

3. when I isolate tissues from rats for my project, can I simply transfer the tissue into centrifuge tube instead of centrifuge tube ?(disruption is processed in a 2 ml centrifuge tube using TissueLyser)

I would like to isolate RNA from plants, is an low cost RNA isolation protocol available? Preferable using Chloroform as main reagent.

Thank you in advance,

Kiriakos Athanasiadis

I found only kits which purify genomic DNA and total RNA sequentially. I'm looking for a protocol for isolation of total RNA and plasmid DNA from the same sample in mammalian cells.

Recently, I did an experiment where RNA was isolated by TRIZOl reagent.

Finally the concentration of RNA check by nanodrop was around 5-16 microgram/microliter.

Their 260/280 ratios were in the range of 1.98-2.1 and also the 260/230 ratios were in the range of 2.1-2.3.

However, unfortunately the quality analysis by qubit 3 fluorimeter analysis showed RIN values less than 3 and were in the range of 1-1.8. I have to do downstream analysis RNASeq.

Gel was also run but only single band was obtained everytime.

I dont understand when concentrations and ratios were in good range then how can I monitor where I am going wrong. Why it failed the QC by Qubit?

I request to help me findout the reason and trouble shoot the problem..

Thanks

RNA isolation

I'm performing qPCR on bovine whole blood samples collected 9/2021-11/2021 and stored/thawed a handful of times before initial PCR testing for anaplasmosis locally and then for BLV via PCR at Michigan State before being stored again and sent back to me at my lab for some experimental testing for coxiella burnetii on beef cattle. Upon receiving the samples on dry ice, I subsequently stored them at -80 C before I took them out for a few hours to thaw before sample preparation and extraction using Magmax Express -96 Deep Well Magnetic Particle Processor was performed. Our lab has typically used 18s as the endogenous control for multiple different tests, but I suppose I'm unfamiliar if the specimen type is having an effect on the Ct values? One of my NA plates had several wells with no Ct value for 18s, so I did a 10:1 dilution and a Ct value for 18s did eventually show, but it was still higher than I'd like it to be (Ct ~26). There is also a decent amount of variation between the Ct values for 18s samples being tested on the same plate. I think I either inherited some samples that have some agent causing PCR inhibition, or the whole blood samples are no longer viable due to several rounds of thawing/freezing and a couple of trips across the country for other labs to carry out testing. I'm following our institutional SOP that is actually designed for coxiella burnetii on small ruminants, but the thermo fisher kit I'm using has a similar protocol including specifics on how to prep and test whole blood, however, my lab director insists that the thermo fisher MagMAX™ -96 Viral RNA Isolation Kit can be used with whole blood samples, even though there are other kits specifically made for specimens with more cells that what we use the viral kit for, which is for testing ruminant fetal tissues.

Any help would be greatly appreciated!!!

This kit is designed for isolation of viral RNA and DNA from cell-free, or mostly cell-free samples. Cell culture medium, swab samples, and biological fluids such as serum, plasma, urine, meconium, and nasal fluids can be used with the kit.

• The MagMAX™-96 Viral RNA Isolation Kit procedure accommodates up to 50-µL sample input, which is sufficient for most applications. Sample volumes up to 300 µL can be processed to increase the detection sensitivity of low titer samples using the MagMAX™ Pathogen RNA/DNA Kit (Cat. no. 4462359).

Hi, I am transfecting mammalian cells with a miRNA mimic. After the 24-48hr transfection incubation I proceed to RNA isolation. My question is must the RT-PCR be run straight after RNA isolation or can the RNA be stored at -80 for some time before proceeding to qPCR and gene expression effects from the miRNA mimic will still be observed?

Hello all, I have been trying 5x Magmax 96 viral RNA isolation kit (AM1836-5) for extraction of total RNA and DNA samples from swab samples (assuming that the viral titer is too low). We have Magmax Express 96 magnetic particle procedure to automate the extraction procedure. So I prepare the plates as per user guide instructions and run the script using the instrument. Please note that I add a spike in RNA control in all of my samples and add two extraction controls in the plate with the spike in control and nuclease-free water instead of the sample to verify whether the extraction went okay. Following extraction, I run conventional PCR using primers for the spike in RNA control. I do quality check using NanoDrop and Qubit and the readings look okay. However, PCR did not work for any of the samples. No peak even for the spike in control in the tape station and Qiaxcel runs. Although PCR positive control worked perfectly and got the expected band size which likely indicates PCR worked okay. There might be something wrong in the extraction procedure which I can't figure out. I tried this process a couple of times with different concentrations of a spike in control (1 and 2 ul in lysis binding solution along with carrier RNA and buffer and isopropanol). Can you please enlighten me a bit about where might be the issue in the extraction process? Thanks a lot for you time and help!

My plant samples are Arabidopsis and maize

plant materials can be of any species.

In the FFPE samples, both DNA and RNA are fragmented. Someone does not use DNase I during RNA isolation of the FFPE sample. They found the DV200 was confusing.

Does DNA contamination affect RNA quality determination using RIN (RIN value or DV200)?

I am new to extracting RNA from fruit flesh. Currently, I'm extracting RNA from sweet cherry flesh using Fruitmate and Nucleospin Takara Bio. I have tried using RNeasy kit Qiagen but I failed (but with another fruit sample I can extract it).

For shredding the tissue I used multibeads shocker and store the sample at -80 degree celsius freezer.

I have tried to follow their instructions and try to create a clean workspace as possible. My problem is that the extraction always resulted in low concentration (both 1x and 2x elution) and absorption is also very small (0.xxx of A260 and A280), also A260/A280 ratio of 1.5 - 1.89. I want to know where did I do wrong but I don't know how to evaluate it. Please help.

My question is how I can increase the quantity of RNA isolated from cells in wound edge in this case from keratinocytes? I have done the experiment and still the quantity of isolated RNA is low for RNA seq. does anyone have experience who could help me?

Best

Before lysing the lung tissue and after putting tissue in RLT buffer for RNA isolation (Qiagen), I realized the tissue lysis machine is not in service.

My lung tissue is on ice in RLT buffer and Im wondering if Im able to store it in the buffer or how I should go about preserving it.

Thanks!

Hi everybody,

I have been doing RNA isolation from human serum, but when measuring the isolate with NanoQuant equipment, the ratio obtained is very low. How the improve the Ratio of RNA isolated from human serum?

I appreciate if you can help me!

I am trying to amplify a gene for a mouse histone linker protein using PCR for cloning purposes. For this, I have used cDNA synthesized from RNA isolated from mouse liver as template, but I am unable to detect any bands after running the PCR samples on an agarose gel. Could anyone please share a suitable protocol for using cDNA as template for PCR cloning? What amount of cDNA should I use per PCR reaction?

Hello!

Last week I found myself researching how different storage of cells in RLT buffer might affect the end results and found many different answers. Some say it is best to snap freeze the cells on dry ice, other say that room temperature is fine. Others mean that storage of cells on dry ice will severely affect the recovered yield. With all those different answers in mind I did a little test and thought id share it with the network.

I used equal amounts of BR5 mouse cells for each sample and then did different isolation methods, namely;

1. collect in lysis buffer, isolate RNA immediately, measure RNA, freeze on dry ice for 1h, measure concentration again

2. collect in lysis buffer, incubate in RT for 2h, isolate RNA & measure

3. collect in lysis buffer, incubate on ice for 2h, isolate RNA & measure

4. collect in lysis buffer, incubate on dry ice for 2h, isolate RNA & measure

I found that there is no significant difference in RNA yield between the treatments, on the short term. So, if you forget your samples in RT, you should be just fine!

Attached is a more detailed presentation of my findings

Best,

Linn

I am planning to do primary macrophage cell culture. I couldn't decide what to use to remove cells from the well plate prior to RNA isolation procedures, for example we usually use trypsin for this. But in primary culture this can be somewhat harmful. What should I use?

Hi everyone,

I work with Arabidopsis thaliana.

I want to know what is the best strategy to strore plant saplings after the treatment to preserve the quality of RNA (at least for a week).

1. Is it adviseable to flash freeze sapling in a microcetrifuge tube in liquid nitrogen and keep it in -80

or

2. Is it better to add RNA lysis buffer, crush the samples and then flash freeze and keep it in -80.

or

3. I should just add the RNA lysis buffer without crushing the saplings?

I know if I flash freeze the saplings without any buffer the ice crystals will still puncture the plant cell, might lead the slow yet possile RNAse activity thereby RNA damage, wherease I think adding RNA lysis buffer will act as inhibitior of that slow RNAse activity during the storage period.

Any other suggestions are most welcome.

I will be happy to get other useful advices that work in your case.

thanks

Hi,

During RNA isolation, why is it required to spin the tubes not more than 10000 rpm rather than 12000 rpm after adding "isopropanol"?

Thanks!

I have an extra kit from Qiagen Cat: 74104, i was curious to know if i can use the same kit for RNA isolation from blood samples. Will it be possible or not ?

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 want to do RNA isolation and RT-PCR of cells seeded on 3d hydrogels with high purity and noninterference of hydrogels. Can you please suggest a detailed protocol to achieve the same?

To perform molecular tests, should we isolate DNA or RNA from tumor tissue and why?

I isolated RNA from the exosome using Total RNA Isolation Kit and stored it directly at -20 C. today I conversted it into cDNA and then ran the qPCR but I didn't get the amplification. I personally think that my RNA may have been degraded as it was stored at -20 C and I added mothing with it. However, various studies and Kits have said that RNA once isolated should firstly be kept in NUCLEASE FREE WATER or ALCOHOL. so kindly tell me as to how to store the RNA properly. and Nuclease free water will be good or the ALCOHOL??? Also, tell me the concentrations as well please.

Hello, I have problems with RNA isolation from gram positive bacteria producing EPS. I obtain a cloud instead of the classic clear rRNA bands. I tried with liquid nitrogen or beads to break cells and then phenol:isoamilic:chloroform or a commercial kit to extract. I always obtain the same result (a cloud in the photo). Does anyone have a protocol for gram positive mucoid bacteria?

Could you recommend any 96-well format RNA isolation kit suitable for human cancer cell lines, organoids and primary hepatocytes, that yields enough RNA for further RT-qPCR?

I need a recommendation for the use of the RNeasy PowerPlant Kit, Qiagen. I have done RNA isolation tests for Macrocystis pyrifera (kelp), nevertheless I have obtained little amount of RNA. The first step has been replaced by the use of liquid nitrogen (for recommendation) but I think that this step may be the problem.

I am trying to isolate RNA from Breast tumor and adjacent normal tissues (stored in RNAlater at -20 C)  using Trizol method. After adding isopropanol , I am getting a transparent droplet like pellet that does not stick to the bottom. And there are no visible bands on gel either. 

Hallo everyone,

I've been having some trouble isolating bacterial RNA from a gram positive organism for a RNA Seq analysis. My problem is that I always get a very intense "cloud band" on the agarose gel around the position where the 5S RNA band should be.. I've tried several protocols and kits, with and without bead beating, Trizol, Lysozyme, but it happens every time.. The first idea was that these are products of degradation, but then again the intensity of the 23S and the 16S bands clearly remains very high. And also, on a Bioanalyzer this 5S band definitely does not look like degradation, but rather as a sharp peak around 127 nt.. Does anyone have any experience with that? If this is in fact the 5S rRNA, why do I get such accumulation, how should I get rid of it and would it temper with my RNA Seq results?

Thank you all in advance!

Hi, I am using 100mg of N. benthamiana leaves for isolating RNA using Trizol. While following the protocol when I add isopropanol, I get a thick white precipitate. How to get rid of this white precipitate?

A protocol for rna isolation from corona virus

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.

Hi there,

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?

Thank you!

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 ?

Dear all,

Which is the best method to isolate tumour organoids from methacrylated collagen hydrogel.

I need to isolate RNA from hydrogel cultured tumour organoids.

Thank you for your help!

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?

Thank you!

The 260/280 ratio is around 1.8-1.9.

Hi,

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?

Thank you!

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?

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.

Need to store cumulus cells for RNA isolation

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!

Hi researchers,

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...

Thanks!

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!

Hello,

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?

Thanks

Milan

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.

Hi all,

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.?

Hi,

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.