RNA Biology - Science topic

Based on the information you have provided, there are a few potential issues that could be affecting your RNA yield. Here are a few things to consider:

In terms of the expected yield of RNA, it's difficult to provide a specific number as it can vary depending on the specific samples and experimental conditions. However, it's not uncommon to obtain yields in the range of a few nanograms to a few hundred nanograms of RNA from IP samples. Troubleshooting low RNA yield can be a challenging process, and it may take some trial and error to identify the specific issue and optimize your protocol. I would suggest experimenting with some of the variables mentioned above and also consulting with other researchers who have experience with the seCLIP methodology to see if they have any additional suggestions.

The large fold-change indicates that the gene is likely not expressed to a high level. Under control conditions the experession can be almost zero, and a slightly larger expression under treatment conditions will result in a very large fold-change.

Low-expressed genes give only low or no counts in RNA seq. It might be that genes with no or very few counts are filtered out from the analysis, because the counts are not reliable. If the gene is not at all detected under control conditions (0 counts in all control samples), it is not possible to calculate any (finite) fold-change at all.

Hi Matt Wu ! Yes, we did and saw the same thing. Please find attached the bioanalyzer results. The RIN is 8.7.

Great question! Did it work? Or did you solve it, develop any protocol? Please share some advice.

Hi Annette, have you tried library preparation from heparin plasma? I am trying this now but unable to solve the problem.

T7 can work very well in the different applications including in situ probe work.

My colleagues use the 9144 kits.

Hi Alexander,

you can try to perform either double mRNA pull down (after the first round of mRNA pull down just do another pull down with new beads' batch - for me Dynabeads from Thermofisher work really well) or you can try ribo-depletion using rRNA depletion/removal kit (e.g. NebNEXT one). I hope that will help you.

Kasia

I hope all government provide more support the research agencies and all researchers particularly health researches.

Erik van Butselaar hi?did it work well for your cell?

Roughly 80% of the total RNA consists of rRNA and so if your treatment causes a decrease in total RNA, then it is highly likely to decrease rRNA synthesis. To a lesser extent, the decrease in RNA content could be due to RNA degradation perhaps via ribophagy. Decreases in rRNA synthesis can be seen easily enough. You can use fibrillarin staining to look at nucleolar size and decrease in nucleolar size constitutes a decrease in rRNA synthesis, as nucleoli are formed on the sites of rRNA synthesis - the rDNA. If you want to quantify the decrease, you can do so using quantitative real time PCR. You can generate primers against the External Transcribed Spacer (ETS) or the Internal Transcribed Spacer (ITS) of the 45s pre-rRNA. The ETS and ITS are spacers that are cleaved during rRNA biogenesis and are degraded subsequently, and therefore a decrease in pre-rRNA would be indicative of decreased rRNA synthesis. You can perform qRT-PCR and use your ETS and/or ITS primers to estimate decreases in rRNA biosynthesis.

This is an old question but for the sake of people searching for this answer, here is the reason:

Nanodrop is just a UV spec reading. It is only looking at absorbance at 260 nm. Plenty of things absorb at 260 nm other than the RNA you make in your IVT reaction. This includes the T7 protein, pyrophosphatase (if you use it), your DNA template, the NTP monomers, the abortives, truncates, and even salt. Your nandrop is giving a signal for ALL of these things.

Qubit is better. It gives a reading based upon an intercalator that is specific for RNA. This means that it will (likely) only give signal for your full length RNA, abortives (above a certain length), and truncates.

This explains the disparity between your nanodrop and Qubit readings. And btw, a reading of 4000 ng/uL on the nanodrop is too high to trust. 10 fold dilute that and try again.

Better than Qubit would be to actually purify your RNA (DNase followed by EtOH precip/dT/etc., ultrafilter, recover) and then run the final material on a gel or capillary electrophoresis or HPLC and then compare against a standard curve.

Pain in the ass, but it's the best way.

The low product during RNA extraction is mainly related to two reasons. First, using low number of cells or less tissue in the extraction which subsequently results in a lower RNA product. This can be easily fixed through using more cells or tissue per reaction.

The second reason which is more frequent than the first one is the inefficient disruption of cell membrane which causes either by inefficient lysis buffer or poor mechanical disruption method. To overcome this, you need to first check the properties of you lysis buffer such as pH, conc., etc and also improve the mechanical way you use to grind the tissue such as longer grinding in liquid nitrogen or use tissue lyser. 

I would also suggest to use proteinase K (~200ug/ml) with your lysis buffer and increase the incubation time for about 3hrs at 50c. This would possibly helps to increase the RNA product without any effect on the gene expression profile.

I think third band is of RNA (28S) because  rather than running on denaturing agarose gel you have run the RNA sample on simple agarose and due to secondary in structures/different oonfirmation in RNA ,more than two band appear. hypothetically if you don't use denaturing agarose gel, more than two band of RNA can appear.

Best wishes

Dear Dr. Maussion:

Thank you for your answer.

I used 400 ul of TRIzol for any well. According to In the instruction of kit , the value of one million cells should be used. It might be diminished cell mount.

Hi,

Your elution volume is probably too high. If your column is not so broad like in the case of Zymo kits, you can try a lesser volume of RNAse free water for a better yield. Also make sure you get rid of ethanol completely before eluting. However, you can also try magnetic bead methods.

Hi Valeria,

Phenol extraction of RNA is always my go to choice if there are problems with column based methods. If you do make your own water or buffer saturated phenol, you will find that to saturate the phenol takes about 10-15% of the aqueous phase. In my experience you will have a similar loss of RNA, particularly with RNA with a lot of secondary structure. I would also recommend a pre-made mix and if you are going to be using enzymes such as reverse transcriptase on the RNA, do at least two chloroform extractions as phenol can be very inhibitory to RT.

I found this kit that maybe could be useful!

Hi,

you may find this link helpful

Good luck

From my knowledge, Syber Green II has affinity to ssDNA  while Syber Green I as affinity to dsDNA. It can stain RNA but with low affinity than DNA.

Thank you very much for the responses, much appreciated.

Thank you Dr. Ernst for the intersting article. I want to see that for how long my RNA is stable in the media and then in vivo such that it does not degrade during transfection process. Further to my concern, I'm now using a buffering solution with chaotropic salts to inhibit any enzyme activity on the RNA. I hope it works!!

Hi Miriam,

We use RNALater for our bacterial culture and keep them on the bench for up to 1 day or at 4*C for 1 week and extract RNA with high yields and good downstream applications. We usually harvest 3-5 mL of bacterial culture and centrifuge at high speed for 2 m, then resuspend in 750 ul RNALater (this process may depend on your bacteria?). When we are ready to extract the RNA, I add my lysis buffer directly to the RNALater + bacteria mixture. I have not noticed any transcriptional change by doing it this way. Hope this helps!

How are you testing DNA contmaination? With wich buffer are you incubating your sample with DNase? Have you tested it against some DNA to see how it stands? You could alternatively buy a RNA isolation kit, like Direct Zol from Zymo. It comes with a Dnase.

Mandeep gill has rightly suggested.

That will depend on your application. If you're doing any NGS application you should use a Qubit that's what most genomics core facilities do before they run samples to make sure samples are accurately concentrated. If doing qPCR it matters slightly less but at the low RNA concentration you're working with Qubit is generally better. I wouldn't pay much attention to what thermofisher or any company claims in a manual regarding limit of detection/quantification as this is almost always exaggerated. 

Also, you probably know this but you can get an idea of the purity of the 9ng/µL you got with the absorbance ratios http://www.nanodrop.com/Library/T042-NanoDrop-Spectrophotometers-Nucleic-Acid-Purity-Ratios.pdf 

Again, all depends on what you're doing. If you're cloning or something none of this really matters, if doing NGS library prep it is very important to get the concentration right. 

The tissue abundance is a critical issue; I had these high Cts for a certain cell type. When I checked on UCSC and tried another tissue where it was seeming highly expressed I got a super thick band on my gel.

So,you might need to do a tissue panel and then to do the RACE where it is indeed expressed - otherwise it is always hard with low abundance templates. Of course, they are not always expressed in the same manner across all tissues. So tissue specific expression should also be kept in mind.  Had satisfying results with both Invitrogen & Takara.

All the best.

Dear Edward, say to your customer that they send a new sample without any extraction using with RNA later solution. I think this way will be better and confidental for you and them. In other confidental way for transporting RNA is working with lyophilised sample. If they have this possibility they can send you lyophilised sample. 

Thank you Nathan. I'll have a go with Trizol. 

To my impression, I think not many reads are mapped to Sox2 gene from your RNA-seq reads. This might cause bias  by insufficient coverage of that gene. From the figure you posted, some part are not well covered. In this case, quantification of the gene by the reads may not be accurate. I would suggest using qRT-PCR targeting different part of the transcript 5' or 3' using your samples (if you still have) to see if the bias is till there. This might complement RNA-seq. If you want to test whether the RNA-seq works well. You may also choose some more abundantly expressed genes, which might have higher coverage by sequencing reads.

You can try using Invitrogen plant Concert RNA reagent . It work on banana which contain very high level of phenolics.

Any process that produces apoptosis will affect RNA one way or another,because during apoptosis more than 500 enzymes are in charge of proteolysis of cell components and also of Ribo and Deoxy  ribonucleic proteín..

you can use RNAlater Solution

Does the large rRNA in this species (26S) have an internal nick or spacer, so that it migrates like 18S rRNA? If so, it may not be degraded, much.

Hi everybody, sorry for my delay, and when asking for the Bioanalyzer image I actually meant the trace – I have seen only now that you have already posted the PDF in the first question.

I agree with Matthew that the library should be of sufficient quality for sequencing, and that 4µg is quite a lot. We usually use 1 µg of high-quality RNA (i.e. directly extracted from cells/tissue). Furthermore, we have recently begun to use only 60% of a reaction to save money, and can still amplify with only 8 PCR cycles.

Tushar, how many PCR cycles did you apply?

To end with the SPRI – Ampure XP are actually SPRI beads, so there are lots of size selections within the Truseq protocols, which is based on the ratio of beads to reaction volume. You might have noticed, that this ratio is different after the ligation step (in order to remove adapter dimers). See also http://core-genomics.blogspot.de/2012/04/how-do-spri-beads-work.html

Of course you can but better if you add 70% alcohol, sample will be safe for long time.

Best

Devesh

Dear Jatin Sharma

One reasons of the capping, protection of mRNA from attack of 5' exonuclease enzymes. Because, remove only one nucleotide from mRNA, produce inefficient protein. Therefore, preservation of mRNA sequence in tranlation is very important. In comparision with mRNA, tRNA and rRNA sequences are not important.

Best, Roshdi

You can use vacuum concentrator at a tempreture of 30 degree celcius for 15min. Place the RNA on ice for 10- 20 min and then return it back to the concentrator for another 15 min. The concentration should substantially increase.

Alternatively, you can preheat the elution buffer at 50-55 degree for like 20 min and immediately use it to elute your RNA. Normally this practice gives me better RNA yeild.

Hope this helps

Bashir

To get best result, it has to be injected in to the cell.

From the publications you sent me:

talks about co-regulated miRNAs, which probably means that there is another factor (e.g. transcription factor) that regulates groups of them in the same way.

indeed uses the wording "Interacting miRNAs" but they do that in the sense of network relation, not physical biological interactions. From your question it sounded like you were interested in the latter.

I do not know about any evidence for direct interaction between mature miRNAs coming from different loci. I can imagine that a miRNA could negatively regulate the host transcript for another miRNA and result in there expression level being reverse correlated. With the positive correlation between miRNAs you should most likely assume a third actor's fault - e.g. transcription factor.

hi saurabh,

I don`t  know any answer about your question.but I have a question .Taqman MicroRNA assay is used in your research .I want to use applied biosystems products .Unfortunately, I have just a few information about them. Could you give me some information about assay ID (382)?Is this assay ID consist of primers and taqman prob(for real time) except of stem loop primer for RT?Thanks.

I'd be interested to see what your 230/260 ratios are, since in my experience cold isopropanol brings down an awful lot of guanidium salts (which TRIzol contains). I always use isoprop at room temperature, where it successfully precipitates RNA without salt co-precipitation, and reserve ice-cold/-20/-80 conditions for ethanol precipitations.

I also wouldn't bother DNAse treating, frankly, though a lot depends on your downstream plans for this RNA. 

Hi Jack M Gallup,

Thank for your reply. i should be aware of this matter in future works.  

The disease state of the RNA sample may not effect you analyses depending on which genes you are looking to use as a control. IF you are doing human work you could order extract from a cancer line already isolated for you and quantified. There are also some qPCR kits that have their own control standards included. Or you can order tissue and extract your own. There are studied transcripts typically used for controls that are well quantified in different tissues, like B-actin.

By the way, forgot one more point, when running a regular TAE gel to check you RNA (so not the usual denaturing gel with formaldehyde and MOPS), it's more convenient to use a 1.5% agarose gel, it keeps the bands more compact and you see better the RNA. And run it short (2-2.5 cm will suffice) and fast (it's single-stranded, although folded, so it will run fine). 200-500 ng are more than enough.

That is precisely it – the omnipresence of RNase on skin and finger prints and on flakes of dust from head and dermis, in sweat, on water droplets from the mouth and nose, etc., hence the need to take barrier precautions.  Essentially to preserve RNA integrity one needs to extract RNA from cells and tissues in the presence of chaotropic denaturing agents – classically phenol and guanidinium thiocyanate – and then to physically separate the RNA from the denatured proteins including RNases that would re-nature on dilution of the denaturing agents.  This was classically achieved by ultracentrifugation through a dense layer of Caesium Chloride, leaving proteins floating above and a 'contact lens' of purified RNA at bottom of the tube. but now much more easily achieved using commercial column-based kits.  Of course, having obtained purified RNA one then needs to avoid reintroducing RNase-bearing contamination, hence the barrier method approach and the use of sterile glassware baked at greater than 160oC to carbonise any existing protein and RNase free-plasticware achieved by a combination of high temperature at which the plastic is moulded and gamma irradiation and not subsequent contaminating the plasticware with sticky fingers!

 As Dr Shapiro says, RNases are proteins, which are not especially stable in the sense that they can be denatured by heat, alkali, chemical modification, etc., but are extremely resilient in terms of their ability to self-assemble (and reassemble following denaturation), actually a property of all proteins in collaboration with the cellular machinery to eliminate incorrectly folded molecules, but enhanced for RNases by the small number of disulphide bonds and thus the smaller member of crosslinks to go wrong.

 Dr Shapiro cautions that RNA may not have come first in the evolutionary process and we certainly need to know much more about molecular evolution to be able to describe an accurate ontogeny.  However, it is clear that if one heats a 'soup' of simple carbon and nitrogen-based chemicals, ribonucleotides will emerge, some of which will polymerise randomly to form short RNA molecules which possess catalytic activity – the original ribozymes – and can promote the assembly or degradation or RNA or amino acids also present in the mix.  You should not overlook that 18S and 28S RNA remain at the core of ribosome protein synthetic machinery today.  At some point, the evolutionary pressure to preserve the 'good experiments' of nature emerging from this soup would have exploited the information coding and transfer capacity of certain RNA molecule combinations 'coding' RNA and 'transfer' RNAs.  However, at some point the information coding role passed to DNA, a double stranded molecule allowing recombination of information and thus the emergence of sexual reproduction.  Meanwhile the chemical stability of RNA leading to the coding signal being 'on' all the time would have been a barrier to the emergence of 'regulation', the answer being to rapidly degrade RNA through enzymatic means to 'turn it off'.  I could go on, but as you begin to appreciate there are books to be written about this and indeed many that have already been written that you should seek out yourself to read.  However, you should not lose sight of the basic facts of RNA, a chemically stable molecule than can be extremely rapidly degraded by enzymes that follow all the rules of protein chemistry but are highly resilient because of the way that they have evolved.

 Hope that helps and I look forward to Dr Shapiro adding his take on the story!

I agree with Saurav the kit can be used for 1pg-1ug total RNA template range to synthesize cDNA. If in pictograms, use ABI pre-amplification kit before PCR; in ng and ug, you do not need pre-amplification before PCR. Check out this paper.

HI Abid,

Yes I am currently collecting references of articles and making a big list and then I try figure out some guidelines. I was just hoping that active researchers in the field might already have some key references available that I could look at. Everyone today speaks of various depths in generalities but finding true papers for human for specific messages types is more difficult but I am making progress. Thank you for taking time to respond to my questions and I wish you all the best in your research.

The RNA Pico assay is very unstable. Any kind of RNAse contamination, chemical contamination, humidity on the backside of the electrode holder, change in salt concentration (1xTE as RNA dissolvent can be already too high salt concentration) can mess up the assay.

If the samples in the same run look okay, you will probably have to replace your ladder.

Dear David,

Concerning the images, I don't understand why you had problems to view them, as the only thing you have to do is click on them and a high-resolution full screen image is popping-up. I just tried and it worked perfectly. There must be some issue with your computer.

Concerning you answer, yes, I avoid I forgot everything about green plants, and that's why I asked this question. Thank you very much for your answer, it makes now complete sense to what we observed, so the additional peaks at a position similar to those of prokaryote rRNAs must be the ones corresponding to leaves organelles, chloroplasts and/or plastids.

Thank you

Thank you so much

Hi all,

Apologies for the radio silence!

It turns out that the homebrew biotinylated probe depletion works a treat (with some caveats).

The steps:

1) Design PCR primers to amplify rDNA genes from genomic DNA. You want to cover basically the whole RNA sequence. For longer species - 18S and 28S (L/R) - we amplified two halves of each gene (so, two probes for 18S and a total of four probes for 28S)

2) In vitro transcribe your rDNA PCR products by incorporating Biotin-UTP into the mix. There are plenty of different ways to do this - choose your favourite

3) Quantify your RNA probe concentration using Qubit and pool your probes so that there is at least a 5x excess of probe:RNA (this requires optimisation, sorry)

4) Hybridise your probe mix to RNA sample, followed by incubation with strepavidin-coated magnetic beads.

5) Keep supernatant! This is your rRNA-depleted sample.

Each step requires optimisation. I found that the IVT (despite being quite a routine procedure) was quite temperamental. You can assess depletion using qPCR against rRNA species - I find this is much more quantitative and informative than Bioanalyzer analysis.

NOTE: you may have to tinker with the probe pooling concentrations. I found that if I add too much excess that the beads don't completely remove the probe and then when you do the qPCR it looks like the rRNA levels have actually increased!

CAVEAT: I had success removing all species but 5S. This is the same as Ribo-Zero. Not something I would lose sleep over as 5S is smaller than most library prep size cutoffs (usually 200nt+). Maybe you will have more success?

However, after all of this, I have all-but-abandoned this protocol for RNA-Seq. We are currently using a kit from NuGen (Ovation RNA-Seq System for Drosophila - http://www.nugen.com/products/ngs/ovation-rna-seq-systems-1-16-model-organisms) that is VERY reasonably priced per reaction and has a nice "rRNA exclusion" step where - rather than depleting - it excludes unwanted RNAs from PCR amplification in the final step. It's very nice, and in our first samples we saw between 1.5-5% rRNA in our final sequencing libraries. Plus, you can customise it to exclude other contaminating RNAs.

If anyone has questions, feel free to shoot me an email at alexandra.mccorkindale@mdc-berlin.de

Have Sen buy you a plane ticket to come out to visit me in Albany and we can use the ddPCR to make the measurements! :-)

Ok, seriously, I would use Trizol, and use phase-lock gel (heavy) to sequester the organic phase away from the RNA when you phase separate to get a nice clean extraction. Then you should have very nice RNA for any downstream use.

But really, design HEX labeled probes for your gene of interest (keep your product size to around 120 bp if possible, and check it in mFold for nasty structures; I have a reference that is FAM labeled), then fly over here and we run the reactions.

Let's get this straight. Pure RNA has an A260/A280 of 2.1. You will see in many protocols that a value of 1.8-2.0 indicates that the RNA is pure. As Robert rightly said, low A260/A230 is solvent contamination and most of the time does not affect downstream applications. It also correlates with quantity so you will find that the more RNA you have then higher this value will be.  

I would stick with Qiagen's columns rather than mess around with the old school Trizol and Chloroform.

This is the best way to pool many libraries. We did similar experiment, where we measured differential gene expression in 14 different mouse tissues using single library prep reaction.

Hi Anna,

Thanks for taking the time to answer my question.

1. Yes, I am aware that RNeasy loses all RNAs < 200 nt. 

2. with lysis buffer I mean RLT indeed

3. I like your suggestion. I can maybe try to take off the slices and put them into the tube and homogenize it with beats. 

Many thanks!

It depends on the organism you study and if you have denature your samples at 70°C during 2min before doing the RNA chip.

For example, if you work with RNA which are coming from some insects, the 28S pic disappear after denaturation... Even if they are eukaryotic organisms, the profiles of yeast will be different of mammalian cells, plants, insects...

Hello,

You must have tried some Trizol based methods, it might be better to try doing it the old CTAB way which works for recalcitrant varieties, here is one protocol which might help.

Do you have only one single sample? Then you can't say much in any case.

If you have other samples: if all profiles look like this, then they will be comparable, so you can calculate valid expression changes between conditions.

If this sample looks typical for one group but the profiles of the other group look different, then you can not sensibly compare the groups.

If this is the only "strange" sample and there are enough other "good" samples, then you can either skip this one (without loosing too much information) or hybridize it and see if the expression profile matches that of the other samples (if not: discard it).

If there are not enough other "good" samples that you must decide on the neccesity: if the samle is utmost precious then I woul use it anyway. If it is relatively simple to get another sample (hoping that it will be better) then I would go for that option.

You won't get information about the reproducibility. I don't know any study that analyzed this. If you have a bunch of samples you can estimate the reproducibility yourself (the confidence intervals [CIs] give you an estimate of the variability). When the sample is behaving strange then the CIs will be wider (but this effect will decrease with increasing sample size). Eventually you must decide if the obtained presicion is good enough for your purpose.

Ok I'll do that.

Thanks for the answers!

Hi,

I do not have experience in extracting RNA from human bile, but I found an article that describes a method for it ("Real-Time PCR-Based Analysis of the Human Bile MicroRNAome Identifies miR-9 as a Potential Diagnostic Biomarker for Biliary Tract Cancer", by Shigehara et al). The authors used phenol-chloroform-isoamyl alcohol and ethanol precipitation combined with a commercially available RNA extraction kit.

Hope this helps.

Kindest regards

Veronica

Firstly, choose multiple shRNAs (at least two) so as to provide specificity to your resultant effects due to knockdown of your target gene. If the target sequence is used before in any other paper, choose that one. If not, then choose three constructs and see the knockdown efficiency. Do check the effect on any non specific protein

Called the Promega tech support for you. Since RQ1 DNase concentration is too low, 5 ul of RQ1 is not the same as 5 ul DNase I in SV kit.  They do not recommend you to substitue.  Sorry for the bad news.  Of course, they would not tell me the concentration of DNase I in SV kit.

Hi Shekhu Ansari

              Initially i also get this kind of band due to protein contamination. After that i standardize to minimize the degradation of RNA. Try to use DEPC treated water for your experiment run in to formaldehyde agarose gel electrophoresis

Hi Wisam,

it is not necessary to buy a product called "DNase removal kit", in most cases the enzyme and buffer is much cheaper if it is not called kit. We are using the DnaseI from ThermoScientific which works fine if the fundamental points of handling DnaseI are followed. Always keep in mind that DNaseI is very instable, so never mix by vortexing but only by slowly pipetting up and down once you added the enzyme. Be careful with storage conditions etc.. I always follow the manufacturers protocol which really works fine in my hands checking by qPCR.

Good luck! 

The

tuberculosis drug streptomycin as a potential cancer therapeutic: inhibition of

miR-21 function by directly targeting its precursor. miR-21 is known as an onco-miR

Sometimes the targets for one miR (like an onco-miR), have roles in other pathological condition. I suggest to focus on targets more. 

In my opinion (and years of experience) the RNA instability scare is greatly over-exaggerated. It is quite stable unless you do silly things like speak or breathe heavily over open tubes or stick your finger in there (not so crazy as it sounds: it's when you take tubes from a previously opened bag - chances are, someone's fingers have been there!). Just use gloves, freshly made miliQ water, and aerosol-barrier tips and you'll be fine. Just saying.

Hi,

In vitro trasncription may generate truncated sequences (affecting the 3' end notably, so the tracRNA). Despite it was shown that truncated gRNA 17-18nt help the on-target (Fu et al 2014), uncontrolled truncated version would lead to more off-target or just non-fonctional sequence. Thus PAGE (and I would add HPLC) purification is the top purification to avoid any risk. Nevertheless, a silica membrane based purification is commonly used and become a real standard. It far is cheaper. And today it has not yet proper tested if PAGE purification actually improve on-target and if on-column purifaction increase the occurence of off-target events.

The case of CAS9 mRNA is a little bit different. The optimal quantity of efficient CAS9 protein seems a key (and the right moment too). So, we should avoid to put too much CAS9 and that is why CAS9 mRNA is preferable to expressing vector. Limited amount of truncated version of CAS9 mRNA would just affect a little bit our estimation of the proper CAS9 mRNA. Thus in silical membrane purification is fair well. Still, I would recommend optimisation of the CAS9 mRNA for the traduction in Mammalian.

DNA is negatively charged, and requires a counterion. Generally, this is sodium, but lithium is sometimes used as it has several advantages: It is slightly antimicrobial, and prevents nucleic acid degradation during freeze thaw. You should be able to use this safely in enzymatic reactions, as I suspect the concentration of lithium would be quite low.

No, Hoechst 33342 is specific for DNA according to

Regards, Christian

Hi

You should first do total RNA or microRNA isolation then depend on your disease of interest you can do different method like:

1- read some paper related to particular disease and find which miRNA is related then run a RT-PCR to detect the miRNA and compare with normal sample.

2-you can use prediction software and predict miRNAs related to gene then use RT-PCR to check expression.

3-you can use micro-array or PCR-array to find different expression pattern in patient and healthy person,also you can do deep seq or RNA-seq.

Best,

Mohsen 

It's a common problem that you don't get good phase separation under high sucrose conditions, and the RNA recovery across different sucrose concentrations (as found across polysome fractions) is variable as well.There's a good description of the associated problems and solutions (including protocols) in the Clancy et al. 2007 Methods paper PMID 17923232. The two main ideas described in there are: (1) add heterologous spike-in RNA to each fraction, which will allow you to correct for differential recovery and (2) first precipitate the RNA from the sucrose-containing fractions with ethanol and then use Trizol on the washed RNA pellets.

You shouldn't have to get rid of the DNAse. Most flow cytometry buffers have 5 mM EDTA in it.  DNAse has an absolute divalent cation dependent activity requirement - there will be virtually no DNAse activity in this buffer - no matter how contaminated.

RBP Map is great, I use it all the time:

You mentioned you run it with DNA? so, what's your goal here? If only study DNA, you can simply add RNase. If you're interested in study sperm RNA, then there're different types of RNA as Jagadish mentioned, then what type of RNA you want to study? that will influence how you extract it, unless you want get total RNA from sperm.

From our experience it has not any effect on miRNA Agilent. 

I have MASSES of experience with Agilent arrays. I find that the easiest way to concentrate RNA is to vauum-dry it (SpeedVac) at ~45oC! It also eliminates the worry about differential precipitation of small and large RNA. In fact, I haven't bothered bringing the RNA to the recommended concentration for years. Instead, I aliquot the right amount of RNA into the wells/tubes, dry to completion, and resuspend in the mastermix with water added to the right final volume.

The reason I started doing it is that at least the miRNA arrays are sensitive to salt, so I was trying to avoid another ethanol precipitation with potential salt carryover.

I have not tried Glycogen, but linear acrylamide does not inhibit anything.

You are likely running into a lower limit on ethanol precipitation. When the overall concentration of DNA or RNA is very low, the efficiency drops exponentially since the nuclei formed are too small to precipitate. The proportion of DNA or RNA recovered in ethanol precipitation is a sigmoidal curve. At low concentrations the yield is very low until one reaches a threshold. To overcome the problem of low yields at low concentrations, a carrier is sometimes added. This fits with your observation that when you double the concentration of RNA you achieve substantially better yields.

GAPDH varies largely under hypoxic conditions. 18s is not very good for comparison of different embryonic tissues. When we design arrays, we use a combination of 5-6 housekeeping genes.

"miRNA primers" is a very ambiguous term. It can mean primers for PCR amplification (SYBR Green- or TaqMan-based), pre-miRNA cloning, sequencing, target-site blocking etc...

What method do you need them for?

Hi Nikhil.

1 million cells is quite a decent amount. I highly recommend using a RNeasy minikit (Qiagen cat no. 74104). I routinely use this kit to isolate RNA from sorted cells. I commonly sort around 100,000 cells and using this kit recover approx 50ng/ul in 12ul (600ng). Using this kit you should be able to significantly increase your yield. The RNA quality is also extremely good so it allows your downstream applications to be extremely efficient. I would steer clear of TRIzol if you haven't used it before as there is a chance that you might loss your tiny pellet of RNA while doing washes, especially if your pellet is clear.

It is common that not all primers designed from the software gave successful amplification. You have to try a couple sets to find the good primers that will amplify your target. Good luck.