RNA Analysis - Science topic

We will collect mouse fecal samples and perform 16S RNA analysis to monitor germ-free status. Looking for any protocol or resource recommendations. Thanks in advance.

I'm in the initial stages of planning a miRNA seq experiment using human cultured cells and decided on TRIzol extraction, Truseq small RNA prep kit, using an illumina HiSeq2500. The illumina webinar suggests 10-20 Million reads for discovery, the QandA support page suggests 2-5M, and I wrote the tech support to ask, who suggested I do up to 100M reads for rare transcripts. Exiqon guide to miRNA discovery manual says there is not really any benefit on going over 5M reads. I was hoping to save money by pooling more samples in a lane, so I was hoping someone with experience might be able to suggest a suitable number of reads.

I am trying to isolate neutrophils from mouse bone marrow with percoll (62%). The goal is to stimualte the isolated neutrophils with LPS and afterwards lyse the cells for RNA analysis. I wonder what is the best temperature to work with during isolation (on ice or room temperature) since I read that neutrophils are not easily responding when put on ice? However working on room temperature comes with a cost of activation during isolation?

I also wonder who is experienced in isolating RNA from these cells?

I have seen many papers and discussions here about various techniques for isolation and culture of microglia and astrocytes from adult mouse brain. Isolation of these cells by Miltenyi magnetic beads is supposed to yield a great purity and have been used for short-term culture (6-24h?) and RNA analysis. Can anyone give me feedback from their experience about separation of cells using Miltenyi beads and culturing these cells for a week (or at least up to 72h) , as described in the Miltenyi protocol?

which RNA should be studied to make a comparison between cells grown in 2D and 3D constructs?

I am separating neuron populations from mouse brains using FACS. I am trying to find a fixation technique for intracellular antigen staining and FACS that will allow long term storage afterwards for RNAseq. I have looked at ZBF which allows long term storage of cells with little change in RNA integrity, but they have to be taken out of the fixation buffer for flow, so RNA integrity will likely be affected during flow and I am not sure if they can be 'refixed' after. For any other fixation methods I have read that mention the shelf life of cells, they refer to how long they can be stored BEFORE staining and FACS, and do not mention any storage AFTER facs.

Hello,

I am going to perform some saliva's RNA analysis. I found in the litterature several protocols to collect and prepare saliva for RNA/DNA analysis. For main of them we have to use kits and there are a bit long. I also saw one using an extraction buffer containingTris HCl EDTA SDS for a whole night at 56°C.

I would like to know if there is a quick and simple protocol to prepare saliva for RNA analysis.

Thanks for your help.

Recently our lab has started a project where we are trying to capture non-coding RNAs (<200nts) by 4SU labeling. This will be my first time working with RNA or doing any sort of labeling. I am trying to find out what step has gone wrong.

I am labeling adherent cells with 1mM 4SU for 5 minutes. I extract using Trizol and then purify either with ethanol precipitation or Qiagen columns. Then I later biotin-HPDP label and pull down with magnetic streptavidin beads. I QC after all my steps and I have RNA going into the beads, but nothing is eluting out with BME or DTT despite various lengths of time/concentration/temperatures. 

I then wondered if the biotin labeling was efficient so I did a dot blot with 4SU labeled and not labeled RNA that both have been exposed to biotin. I see faint dots for all samples at 1ug concentrations, but nothing lower than that so it might be residue biotin and not actually labeling. I then went to see if the 4SU is actually being incorporated by use of nanodrop, but I don't see a peak at 330nm. I'm not sure if there just isn't enough 4SU labeled RNA out of the total RNA (I have tons of RNA). 

Can anybody see where I might have gone wrong or what I can do to fix this? Any more QCs I can do to try to figure it out? 

I am starting a new RNA-seq based project. I will be looking at both coding, and non coding RNAs. To enable me to look at both of these RNA species simultaneously, I will be ribo depleting the samples to remove rRNA. My question is will my RNA-seq data have enough coverage if I want to look at splicing changes. I realise when people usually do this sort of analysis they Poly A enrich samples for mRNAs. I could split the samples in two and do both ribo depletion and poly A enrichment independently but this would cost double for RNA seq. Any help or ideas much appreciated.

I'm a molecular biologist, and i have a few projects coming up in transcriptomes and small RNA analysis. Can i get by without knowing any programming using user-friendly software such an Geneious Prime or another program you can suggest or is it absolutely a must?

RNA Analysis

I performed agarose gel (1%) for PCR products of COX-2 from saliva samples of patients suffering from periodontitis. I could not see any bands and therefore in order to check if the RNA was isolated appropriately, I also performed agarose gel electrophoresis and again could not see even a band of RNA. Can anyone suggest where we are making mistake? 

I'm currently investigating a lncRNA and i'm very curious to see what the subcellular location is due to silencing strategy and prediction of lncRNA function. Could i somehow separate the nucleus from cytoplasm and isolate/qPCR to find out where expression is present?

I was looking at fractioning protocols, but these are mostly aimed at proteins, if at all possible, could you recommend me a protocol for RNA analysis?

Huge thanks in advance!

I am doing RNA immunoprecipitation, using the RNA TRAP technology, with which you can capture actively translating ribosomes with the L10 ribosomal subunit fused with GFP, using GFP antibody coated magnetic beads. Right now I'm testing it on HEK293T cells transfected with L10-GFP and I am having trouble finding good reference genes / controls for qPCR.

GADPH & b-actin seem to be differentially expressed between the IP & the unbound (the supernatant of the IP) fractions, which result in very different & unreliable results when I look at enrichment of GFP between IP & unbound. For example, b-actin sometimes shows Ct value difference of 5 between IP & unbound fraction, of the sample.. Actually b-actin seems to be 'enriched' in the IP sample (so I suspect it binds to the beads..)

What do you suggest I do? Should I compare with the input sample? n

We are doing a pilot study and are stuck on the workflow needed. The starting material is plant and fungi material in RNAlater and end goal is making libraries with the Illumina Nextera Flex.

We are doing the RNA extraction with the Qiagen RNeasy Plant mini kit. We have a couple of options in mind for the first strand cDNA synthesis but which one is best is probably dependent on how we'd get second strand cDNA.

So in short how do we go from RNA to second strand cDNA that we can use for library prep?

Also at what point do you do quality control and how? We have most machines available to us (Bioanalyser, Qubit, Experion) but no kits for RNA analyses. Ideally we'd want to keep costs as low as possible.

Hi,

I am trying to purify synaptoneurosomes for RNA analysis from 4-week mouse brains. I am using a Percoll density gradient followed by washing 2X in buffer to remove residual Percoll (Dunkley et al., 2008). From the pellet, I have tried Trizol extraction as well as extraction using RNeasy Mini kit, and neither have been working. Does anyone have any tips or tricks for RNA extraction from synaptoneurosomes?

Thanks!

So I've been doing research on PBMCs isolated from buffy coat, infecting them and then extracting RNA. I've been getting very low amounts of RNA (maximim I've had is about 16ng/ul, but mainly it is below 5) despite using up to 10 million cells per well and then checking the number of cells on the plate by the microscope to check they've been removed. Does anyone have any ideas on how to increase the RNA yield? Myself and 2 previous phd students have found the same. Thank you!

I've used both the Qiagen RNA extraction kit and the Nucleospin RNA extraction kit

Dear colleages

We have carried out a species identification by 16S RNA analysis, and the result is indicating a coccus bacterium, yet our microscopic observation shows that the strain is a bacillus (we have repeated the sequencing 2 times).. We are wondering whether there are reported examples of such discrepancy? Many thank in advance.

Since MOPS is one type of Good buffers, can it be substituted with any other? Can I be using TAE/TBE for RNA gels?

My study is on using microbial DNA present on fingerprints as identity markers of humans. In this case, how high is the accuracy of identifying the phylogenetic DNA of bacteria present on a fingerprint with the use of crude DNA extraction? I am only familiar with the protocol based on other papers - how do I acquire enough DNA for PCR

I would like to do neurite outgrowth analysis and live-dead viability assays with Calcein/Ethidium Homodimer-1 on PC-12 cells. I would like to know if anybody has tried these stainings prior to RNA isolation with TRIzol reagent. I seed my cells on a dark-colored scaffold, therefore I'm planning to use Calcein-AM to make visible the neurite structures while neurite length quantifications.

 I am wondering if it is possible to isolate RNA after calcein staining and if the dyes cause decrease in the RNA quality/stability or puts them under stress (so that the RNA profiles of the target gene would be disturbed). I'd be glad to get any comments. Thanks.

Hello,

I am just learning about PCR-DGGE to identify microbes in food samples. I am not from microbiology background, so I have difficulty in understanding some points.

I have some doubts when it comes to interpreting the results.

For example, in ice wine sample

KT323272 Penicillium sp. is identified - in RNA and + in DNA

and

JX681079 Coniothyrium cereale is identified + in RNA and - in DNA

The paper I am reading now is Novel insights into microbial community dynamics during the fermentation of Central European ice wine. The pdf is attached here.

I hope some one can help in understanding this concept regarding DGGE.

Thank you in advance

Hi,

We are looking for some advice on improving our method of detection of Mycobacterium species from fish tissues. We have fish tissues with granulomas and can see presence of acid-fast bacteria in the granulomas. We can extra total DNA and but when we run a Eubacterial PCR we get either very faint bands or nothing. I wondered if the problem was inhibitors at some stage since we were working with formalism and alcohol fixed tissue but when I add in Mycobacterial DNA to the recovered total DNA (ie essentially spike the sample with a known concentration of high quality bacterial DNA) we get a nice positive band. This makes me think that the assay itself is working but we are unable to detect any Mycobacteria in our sample either because its not mycobacteria (possible but unlikely given the pathology) or its at such low levels we are unable to get a quality result. I was hoping to send the PCR product for 16S r RNA analysis to confirm if we have Mycobacterium but am struggling. I have done this successful with Gram negative bacteria including from formalin fixed wax embedded tissue sections but really struggling with the Myco - any suggestions gratefully received!

I have a RNA sample with 300 ng/uL of concentration and 260/280 as well as 260/230 ratios higher than 1.9. I ran an agarose gel to test the quality of my RNA sample and my loading buffer had a denaturing agent formamide. I followed the protocol mentioned in one of the articles about RNA electrophoresis. It says that I can run my RNA on agarose in normal TAE provided I have formamide in my loading buffer. However, I do not see any sort of band which might correspond to my RNA nor anything in the wells. What problem could this be that I am not being able to understand?

Hi!

I have mRNA after in vitro transcription. Nanodrop shows conc of 4150 ng/ul, (measured at 10x dilution, 415ng/ul), but Qubit and Tapestation show 10 times lower concentrations. The gel from Tapestation shows very weak band. I do phenol chloroform purification after IVT which is suppossed to remove most of the free nucleotides. The A260/280 ratio is 2,05 and A260/230 ratio is 2,34. I have no idea where does this difference come from. I tried different Nanodrops and different Qubits...

Maybe you can help?

Hello everyone.

Right now i am working on differential expression of miRNA using next generation sequencing. analysis post sequencing was done using "Small rna analysis" pipeline in a integrated analysis software. I have disagreement with my supervisor regarding the results of dysregulated miRNA in terms of normalization method used. I use small rna analysis pipeline, because it was explained in the software about this pipeline if i want to analyse miRNA differential expression. Many journals also use this pipeline to find differential expression of miRNA. Regarding normalization method, i also read that method of normalization for RNA seq can be used for small RNA seq, for example DESeq/edgeR, TMM etc. but my supervisor need more assurance because he wanted normalization method specifically for miRNA sequencing. Since i am new in this field, i need some confirmation from experienced people in this forum.

1. Is it right for me to think that miRNA sequencing is the same as small RNA sequencing, and almost the same with RNA sequencing (just different on enriched small RNA annotation)? if

2. if previous question's answer is yes, so is it okay to use normalization method usually done for RNA seq,for example DESeq etc, and what is the reason for this?

if the answer is no, so what is actually the difference between those sequencing, and what kind of normalization can be used specifically for mirNA sequencing?

Looking forward for any link or elaboration from experienced people here in this forum.

Regards,

I want to make some single-cell RT-qPCR or scRNA-Seq experiments. But, I'm affraid, that during tissue dissociatation and FACS sorting are cells stressed, and it's activate expression of some genes, which we want to study.

So, in my idea, I want to first make tissue fixation, after that dissociate cells and continue with sorting/analysis. Have anybody experience with protocol like this?

Hi,

I want to isolate exosomes for RNA analysis from blood serum. Blood samples take about 5 hours to reach the laboratory. İn which conditions can I transport blood samples properly????

Hello All,

This is the image of Total RNA non- denaturing gel electrophoresis. The 2 rRNA bands (28s and 18s) are prominent with 2:1 intensity but there is no smearing in between them that indicates presence of mRNA, so was the mRNA lost during extraction or what could be the possible reason for the disappearance of mRNA? Can I proceed the remaining Total RNA extract with In-vitro Transcription?

Thankyou.

Hi everyone,

We are planning a RNA analysis of expression of mRNA of an interest protein. The primers that appear to be the most reliable are Taqman primers, suggested to work better with Taqman Polymerase Mastermix.

However, we already have a master mix from Agilent (PfuUltra II HS DNA polymerase). Has anyone tried using the Taqman primers with other polymerase types with success? We are afraid to ask the manufacturers as, of course, they will prefer us to buy their own polymerase.

Thank you in advance.

I am having a bit of trouble trying to obtain good quality and decent amounts of mRNA from ~5,000-10,000 FACS sorted cells.

Tumours are being dissociated to a single cell suspension, stained for the markers of choice, and sorted by FACS in to media containing 20% FCS and spun down. I've also tried sorting in to lysis buffer directly. Using the QIAGEN RNeasy micro plus kit I get low yields and fairly poor quality RNA. Same goes for TriZOL LS.

I seem to be fine with cell lines which have not been sorted. But I fail to see why FACS sorting would change things so dramatically.

Any suggestions very welcome! Thanks.

hello i am trying to do  qPCR for a specific microrna from a virus and i want to confirm the mature microrna and also the pre-microrna. so i want to know how to designer primer to amplify the pre-microrna?

Hi, 

I extracted RNA from Penicillium infected plum and nectarine tissue using the Qiagen RNeasy Plant Mini Kit. Concentrations from nectarine samples were good (67.6 - >200ng/ul using Qubit Fluorometer) but low for sample from plum (4.4 - 34.6ng/ul). I presume phenolics were the problem since the darker (red) the tissue samples the lower the concentrations.

Would it be worth sending the low concentrations for analysis on the Bioanalyzer or Experion or will it just be a waste of money? The budget is tight and another kit to counter phenolics/sugar/salts will add to costs. I can load maximum 66ng RNA from the lowest concentration for cDNA synthesis. I will probably end up loading 50ng for all the plum samples. Would that be okay considering we need to publish? 

Thanks for your input,

Pieter

I grew up bacterial isolates, then did DNA extraction using spin kit for soil, which I have used many times.

This time I appear to have got more bands than just the genomic DNA, why would I have got this and what can I do?

I think there was a lot of starting bacteria scraped off the plates, the nano drop showed an error message so I couldn't get readings but I suspect they are high.

Thanks

My problem is that i'm trying to precipitate RNA with a Ethanol RNA Precipitation protocol and the yield is bad (almost half of initial concentration and half 260/280 purity). My initial volume is 30 uL and 70 ng/ul of concentration approximately and the precipitation gives me worst concentration and purity than the initial one. Is there a minimum volume for RNA precipitation ? because i'm using the entire 30 uL for the precipitation protocol.

Thanks

Any advice on sonicators v homegenisers and brands/types that will get the highest and purest RNA concentrations? My old lab used the diagenode biorupter (sonicator) which worked really well, but it is a bit out of our price range. We have a hand held homegeniser we use for protein extraction/isolation but I'm worried that too much of the tissue is lost in this process for reliable RNA analysis, and I'm wondering what other groups might use? 

We will be primarily working on rat spinal cord/brain tissue.

Any wisdom would be appreciated!

Hello, 

I am sorting monocytes from spleen for RNAseq. I pre-purify my cells using Miltenyi isolation kits and then I FACS sort the cells to obtain a high purity. Since I had problems with the RIN values (below 8) I added RNAse inhibitors to the sorting buffer and the recollection medium since the spleen contains a lot of RNAses. After this I increased the integrity of my RNA to reach values over 8 but not in all samples. Also I directly isolate the RNA after the sorting.

Does anybody knows how to further improve the quality of my samples? is the perfusion of  the mouse with PBS with RNAse inhibitor a good option? it would be toxic for the cells?

Thank you in advance for your help.

Hi all,

Looking for some advice.  I have a small number of iPSC-derived oligodendrocytes (approx 10,000 cells/sample) that I'd like to do RNAseq and whole genome wide methylation analysis (e.g. Illumina 850k).  Can anyone give me any insight as to whether this is feasible?

Does anyone have any experience of doing such work on small number of cells (and perhaps can recommend kits for extraction) and/or amplification steps if required?

Many thanks for any advice,

Vicky

Recently, I am working with a newly isolated yeast RNA for RNA sequencing. I have sent my RNA samples for RIN analysis using Agilent Bioanalyzer. However, I have received error results for all 12 samples. Based on the results attached, the 18s/28s ratio and RIN cannot be calculated. The retention time for 18s/28s for all samples were not within the start time/end time mentioned in the report. Is that why the RNA ratio and RIN cannot be calculated? What are your suggestion/troubleshooting for these results. Is the manually adapted results accurate? Can I use this results as reference for RNA sequencing

I have RNA extraction from mouse kidneys. I had very good RIN values for that RNA extractions (around 9.0). Since I had bad experience with bacterial RNA contaminations I decided to measure the RNA conc. (and RIN) on the same Agilent Tape Station, but chose the "prokaryotic programm" instead the usual eukaryotic detection.

Now my question is if anybody ever tried that and if so would be open share his/her conclusion from that? Is that a possibility to check contaminations at all? Otherwise I don't know how else I could test my samples for bacterial contaminations.

suppose, say that purity of the control sample ( mRNA) is 2.10/1.06, how do you determine its purity? or, How do you know its impure?  

My mRNA concentration falls around

control- 3809.2ul

Injury - 8424.6 ul

 I have been told that differences are high, and  I know it's a  simple math, but what is the optimum difference, or what should be the optimum concentration? 

thank you for helping. 

Hi all,

I'm working on T7 in vitro transcription. I've tried both Neb and Megascript kit, incubated at 37 degree for 2 hours, denatured RNA at 70 degree 5min, then run simply using agarose gel (I'm trying for denaturing gel now).

You could see the attached picture, lane 1 is positive ctrl (linearized plasmid); and lane 2 is my sequence of interest (pcr template).

You could see there are both strong lower bands around ~200bp; while expected full-length bands (1-2kb) are quite faint. I basically could rule out RNA degradation because RNase inhibitor is included in IVT reaction and RNase-away used in gel running and i'm very careful for any possible contamination.

I think it's more likely INT is not complete with T7 polymerase stop around 200bp. So what should I do? I heard people would first unwind/anneal double-strand DNA at 70 degree prior to add polymerase, but is it really helpful and necessary given the protocol of neither kit mentions this step?

Thanks

=================================

Problem solved.

Simply run denaturing gels, either agarose or polyacrylamide.

This is a lesson that RNA will form structures and hugely distort the size on native gel.

thx

I am looking for institution where miR-16, miR-132, miR-146a, miR-155, and miR-223 can be analysed. Some kind of colaboration or cooperation is possible. Please, answer who is working in this field.

I have some DNA samples for genotyping-by-sequence (GBS) extracted with the Qiagen blood and tissue kit without treated by RNase A. I've quantified DNA concentration with florescent dye (Promega Quantiflour). This dye is dsDNA specific with minimal binding to ssDNA, RNA and protein. However, the gel image showed that there may be RNA contamination in the DNA samples. Would the presence of RNA affect GBS in this case? If so, how?

Most institutions require that DNA samples submitted for GBS to be pre-treated for RNA, which suggests to me that the presence of RNA in the DNA sample is bad for GBS, but I do not understand why is it bad (given that DNA concentration can be accurately quantified even with RNA contamination).

Many thanks ahead for any insight!

Actually I'm trying to detect the new subtypes of Influenza A in bats ( H17N10 and H18N11) of the floodable savannas of Colombia.

I would like to know a recommended RNA extraction protocol for samples taking in RNAlater, we have had used RNeasy mini kit by with low yields in the measuring of RNA quantity and quality in NanoDrop.

Someone has experience in the subject or knows the sequences of primers and probes for real-time PCR detection?

I work with RNA on the Bio-Rad Experion chip gel electrophoresis. Is it possible to determine strand length from the concentration results? I have noticed that other papers using this or Agilent don't usually report bp. Is this over reach when reporting total RNA quantity and RIN?

 These samples are showing very good quality on gel exhibiting two intact RNA bands, without any degradation; but RIN value is not good.

We are using tape-station for RIN value determination. Is this platform suitable for plant tissue, or bio analyzer is better for plant RNA?

Kindly help me to know why this is happening?

Upon using the TruSeq Stranded mRNA LT kit for RNA-Seq sample preparation I obtain the following Bioanalyzer traces using a Agilent DNA 100 Series II kit.

What is the additional trailing peak near the upper marker? Will it hamper sequencing?

How many hearts from newborn mouse are needed for RNA-sequencing?

Hi All,

I have datasets derived from RNAseq experiments,I did differential expression for coding and lncoding RNA. I want to construct a co-expression network of these coding-noncoding genes,what should I do? Can you recommend friendly software? I found  WGCNA package of R, but please give me some information about Process .

I should construct a co-expression network for each experiment OR I can select special genes and lncRNA from all of experiments(I mean after doing meta analysis ,set filter and choose key genes) for constructing a co-expression network?

Dear Scientist,

I badly need someone's suggestion who are working with RNA from etiolated arabidopsis. In my case I am working with 4day old etiolated Arabidopsis seedling to isolate RNA by using TriZol reagent.(<100mg/1mL

Here is the condition I am using:(<100mg/1mL trizol, 15 min incubation>200ul chloroform/1ml trizol> 15 min

1. 100mg/1mL trizol, 15 min incubation

2. 200ul chloroform/1ml trizol

3. 15 min centrifuse@12000rpm @-4CAdd 500ul isopropanol> incubate 15 min @room temp. 

4. Add 500ul isopropanol incubate 15 min @room temp. 

5. incubate 15 min @room temp. 

6. 10 min centrifuse @10000rpm

7.Wash pellet twice with 70% ethanol.

But this case I cant see any pellet... :(

Please I need your valuable suggestion to trouble shoot it out.

I've extracted RNA from plant tissues, and want to store it at -20 degree. Is it possible?

I am dealing with Candida RNA isolation from TRI reagent (used qiazen kit for extraction).I am really concerned about the quality of my RNA. According to Nanodrop reading my yields were ~100 to 150ng/ul. The A260/280s were ~2.1-2.2. Although I have used glass beads and vortexed vigorously, the quantity of RNA was not good. I am planning to use SAB (Sodium acetate buffer) for cell suspension and Phenol:Chloroform: isoamyl alcohol instead of TRI. Has anyone experienced on Candida RNA isolation without mechanical disruption? Please suggest me which one is better?

Thanks in advance 

Hi I am depleting the ribosomal RNA of my samples with ribominus kit for ion proton libraries. After running a pico chip in the bioanalyzer I see a clear depletion of the main 18s and 28s (just look at sample 3). However in the bioanalyzer "gel" image I have a very clear low weight black band. I dont know if it should look like that or just not bands. Also I dont know if the black band correspond to the 5s....

Thanks in advance

Dear all,

I am currently trying to find the concentration of RNA present in a solution in terms of copies of RNA/volume of solution. The RNA molecules are 508 bases in size. I will be using the formula involving Avogadro's constant and the concentration of RNA in terms of ng/ul to find the concentration in terms of copies of RNA/volume of solution. The RNA strands are all + sense strands. Although the RNA molecules are not complementary, I'm not sure if they could potentially form regions of dsRNA.  

How can I know if my RNA is single stranded or double stranded in solution? This is important since dsRNA would require me to multiply the molar mass of the ssRNA by 2, which can cause the concentration in copies of RNA/volume of solution to differ greatly. 

Thank you!

Is there a way to analyse the data from an environmental sample at once, I mean prokaryotic and eukaryotic RNA at the same time, or do I need to run the samples on two different chips to obtain the respective quality?

I am analysing an anaerobic reactor sample containing bacteria, archaea and anaerobic fungi.

Hello,

I will be using this kit provided by Qiagen for isolation of viral RNA from stool samples. I was wondering if anyone had any input on how to tweak the protocol to allow for efficient isolation of RNA as opposed to DNA.

Thank you for your contribution!

Regards,

Kyle

Dear colleagues,

I am working on a laser capture microdissection (LCM) project to collect vascular cells from Arabidopsis cotyledons for RNA sequencing. RNA of vascular cells obtained from LCM has been isolated by PicoPure, and we are planning to linear amplify the RNA from nano grams to micrograms for sequecing by 2 rounds of RioAmp HS Plus. I have a couple of questions about the assessment of starting RNA quality and quantity.

We are currently not feasible to use a Bioanalyzer or fluorometry (RiboGreen) so we tried Nanodrop. The concentration was estimated from 2-16 ng/ul varied from different samples. I am aware that the readings below 10ng/ul is not very reliable so I am planning to perform a qRT-PCR to assess the quantity and quality together.

Publications (eg., Kube et al (2007). BMC Mol. Biol.) and kit manuals (eg., Arcturus Paradise Plus Reagent System for FFPE tissues) has introduced the procedures of qRT-PCR for this purposes. Basically the ratio of the RNA yield obtained from different regions is used as an indication of RNA quality, and the RNA quantity derived from the 3’ primer set is used as the quantity measurement of the RNA, by plotting standard curve of control RNA amount vs. Ct. This assessment is based on the assumption that the actin mRNA in the sample represents the average status of other RNA molecules in the same sample. The total estimated RNA amount in a given sample is expressed as an equivalent of universal RNA that contains the same amount of actin mRNA. Most of these protocols are based on using ß-actin in human, would you think something like Actin2 would fit this assumption in Arabidopsis (plant)?

Actually, I have done a semi qRT-PCR by using a series of dilution of a control RNA (isolated from fresh frozen samples) whose quantity and quality has been assessed. See the figure attached.

Apparently, the nanodrop estimated RNA concentration (and therefore the input) is not consistent with the band intensity. Is this because:

Any comments will be appreciated.

Cheers, Thomas

When we run total RNA samples on a 10% TBE Urea gel followed by SYBR Gold stain, we can easily tell the 5.8S rRNA and the 5S rRNA bands. We normally call the multiple bands below the 5S rRNA band tRNAs bands. Normally there are two weak upper bands (Band 1 & Band 2) and one super strong lower band (Band 3). I assume the strong lower band (Band 3) is where most of the tRNAs locate, around 70-75 bp. Does anybody have any idea what are the two weak upper bands (Band 1 &2), especially the one right above the lower strong band (Band 2)? Longer tRNAs? tRNAs precursors? Or tRNAs with unspliced intron?

I want to do research to compare mRNA in two groups and want to calculate the sample size for the study. Most of the articles has given expression of mRNA as fold change. Please help me how can i use these values, and what will be the formula.

I have been doing TriZol RNA extractions of zebrafish embryos with and without glycogen as a carrier. I've found that using glycogen during the isopropanol precipitation step increases the size and visibility of my RNA pellet, but I am also getting a large peak in the 220-230 nm range in my NanoDrop spectrograph. My samples without glycogen look great.

I know that peaks around 230 nm indicate contamination (phenol, chloroform, protein, etc.), but it doesn't make any sense to me that only adding glycogen would cause this contamination.

I've attached graphs from some of my NanoDrop readings. The green line is my glycogen-free sample, the rest have glycogen in them.

I need to store a single cell after isolation for future DNA/RNA analysis.

I did a RNA isolation with a Polysome Pulldown + TriZol, followed by a purification with a RNeasy Kit. With the isolated RNA a transcriptome should be generated.

My samples on the Nanodrop looked quite clean.

(Exemplary sample value for 260/280 is 2,19 and for 260/230 is 2,48. I know that the 260/230 is a bit high but I couldn't find an explanation why. Also it shouldn't be a problem for generating the transcriptome I was told.)

Now I got the Quality control data, which were measured with a Agilent 2100 Bioanalyzer and they look quite strange. 

18s/28s rRNA bands cannot be clearly descriminated, although the sample itself doesn't look degraded. You don't see a smear but clear distinct peaks. See attached an example.

Does anyone has an explanation for this?

Also an additional note: If I digest my sample with RNases, I cannot find any bands afterwards. So it seems those bands are RNA and not DNA contamination or whatsoever.

Don't know if it is important but those are c.elegans samples.

Also the all the sample look quite the same (n=6). You can't see a big difference between days.

Has someone experienced this before or does know what happened in my samples?

I am happy about every help I can get. :) 

I'd like to know the features of single stranded RNA molecules such as diameter and charge, but I can't find any experiment on the subject. There are some works on dsRNA and hairpins, but nothing on ssRNA; I know it is a very difficult experimental matter, but I find strange that there is nothing at all about it.

I am planning to perform RNA seq using a MiSeq Reagent Kit v3 600 cycle, mean insert size of ~600bp, 2x 300bp reads, paired-end. I have had some divergent advice regarding the no. of reads required per sample to perform differential expression analysis on mammalian cells engineered to produce very large quantities of therapeutic product. 

How many reads will I need to allow me to perform differential expression? Does the length of my read or the fact that they are paired-end reads effect the no reads required per sample. I have been advised that 5 million reads per sample or 15 million reads per sample are suitable depending on the expert that I speak to. 

Any advice would be much appreciated.

Thanks

Clair

After a few practice runs on extra samples, I was achieving a 260/280 of about 1.9, a 260/230 of about 1.6, and a yield of about 80ug.  These were not perfect results, but I felt confident enough to begin isolating RNA from my real samples, and after the first run with the real samples, the 260/280 came out at a respectable 1.8 average, but the 260/230 hovered around 0.6 and the yield around 35ug. 

While I was performing the extraction, I felt that this was my best set until the nandrop results came back.  I am at a loss as to why the results were much lower than expected and would appreciate some feedback before I move on to isolate the rest of my samples.

I used a Qiagen kit to isolate RNA from human fat samples ranging anywhere from 5mg to 100mg in size.  I noticed no strong correlation between amount of sample and yield; however, I did notice that overall the samples were much 'uglier' than the extra samples I practiced on (meaning they were much more bloody and something about them was off).  I made sure to homogenize very effectively, I did not grab any interphase layer (and made sure to leave some supernatant in the tubes so as to not disturb the interphase layer, and I followed the protocol to the dot.  Oddly, one of the samples returned respectable results in the nandrop analysis (260/280 of 2.01, 260/230 of 1.64, but the lowest yield of about 27).  I doubt that I ran every sample incorrectly except this one, so that leads me to believe that my samples were not good in the first place?  

I am thinking about extracting the RNA from the column with a much larger quantity of H20 than the perscribed 40 uL, then speed-vac to concentrate, as this will yield more RNA and evaporate residual ethanol to possibly improve the 260/230, but I have to clear this with my PI first.

Any thoughts would be nice.  

Thanks!

Hello everyone, I´m using the Agilent's Small RNA chip for detection of microRNA, but I´m not sure of the indicated concentration by the bioanalyzer (18% of miRNA), because I didn't observe the "hill like" pattern showed on the manuals (fig 1), instead I observe "peaks" (fig 2) on the miRNA region  (from 15 to 40 nt). Is that enough evidence of the presence of miRNA in the sample?

I have been doing RNA-EMSA with Biotinylated-RNA. The problem is that the complex band always show in the side of the lane, especially in the low concentration of protein. Does anyone know the ways to fix this problem?

Here is brief condition of my experiment: My protein is 55 kDa with histidine-tag. Predicted pI of the protein is 8.85. My RNA is 174 bases. Binding buffer contain 50 mM Tris pH 7.6, 10 mM Mg(OAc)2, 65 mM NH4OAc, 1 mM EDTA, 0.2 mM ADPNP, and 5% glycerol. The gel is 5% non-denaturing polyacrylamide gel (with 5% glycerol) in 1X TBE. Run at 4C at 120V for 2h (pre-run 30 min 80V). I also tried tris-glycine buffer which had similar result.

If you need more information, just ask me. I will answer as soon as I can.

Thanks.

Hello, I ‘am looking for information about bacterial DNA purity from Magna Pure 96. I will use the MP96 for purification of bacterial DNA for 16S RNA analysis on Illuminas MiSeq. The purity of DNA has a very big influence on the results of the NGS. Does anyone have experience with MP96 and Illuminas 16S NGS? Has anyone compared the purity of bacterial DNA from the MP96 with other instruments?

Thanks!

Olaf

Actually i have done RNA sequencing for my experimental purpose where they shows how much genes got up regulated by conversion the data through Log2 conversion by comparing with control and drug treatment sample but my question is that is possible to Calculate the P value and FDR value  from this data format here below i have attach a Picture.  if possible  how could i do this any instruction or procedure will be great helpful for my research 

Has anyone seen Total RNA profiles like this? There is degradation but also some other contaminant. The lab told us that these are from mouse colon and bacterial RNA can be a contaminant. They repeated RNA extraction and got similar profiles. Tissue was frozen on dry ice and wasn't subjected to free-thaw. However, it's clear that these are partially degraded. Could the spikes be due to bacterial RNA contamination? Thank you for your input.

I am using the TriZol method for extracting total RNA from adult zebrafish tissue, whole Xenopus larvae and whole zebrafish larvae. Considering that my 260/280 and 260/230 values are near about the prescribed values, do I still need to do a DNAse treatment. Some have suggested that RNA extracted from tissue can never really be genomic DNA free. Any advice, please?