Shortly:

Add 1/10 vol of NH4OAc, en 2.5 volume 100% cold EtOH (100%) and 1 µl glycogen (if you think you'll the pellet without see glycogen you can do without it)

mix

Incubate 30 min at -80°C

Centrifuge 20 min at 12000g at 4°C

(remove supernatant)

Wash with 75% ice cold EtOH

Centrifuge 5 min at 12000 g at 4°C

(remove SN)

Re-suspend the pellet in DEPC (can take some time)

we use it and it works.

Hi, do you still to go on with your Trizol extraction or is a change in the etraction method also an option? Cheers, Nadine

When extracting RNA from skin samples, you can add a Proteinase K digestion step prior to the Trizol extraction, it will increase your total RNA yield. Adding a further ethanol 75% wash at the end of the extraction will also improve a bit the overall quality of your sample, regarding the absorbance ratio.

Additionaly, check the articles by Donald Rio et al. at CSH protocols, they have useful tips and guidelines.

http://cshprotocols.cshlp.org/cgi/content/abstract/2010/6/pdb.prot5439

You could also consider using specialized kits for this kind of extraction, like the RNeasy Fibrous Tissue kit from qiagen.

http://www.qiagen.com/products/rnastabilizationpurification/rneasysystem/rneasyfibroustissuemini.aspx#Tabs=t0

Furthermore, in this report by Bruning and co-workers, they use a liquid N/mortar-and-pestle extraction with qiazol (similar acidic-buffered phenol from qiagen) followed by a cleanup step. Since they use their samples for sensitive downstream applications, could be worth to consider their method.

http://www.biomedcentral.com/content/pdf/1756-0500-4-438.pdf

To remove phenol, do a step of chloroform extraction. It easy and efficient

To remove olysacharide this is more time comsuming . The classical method is to use GuSCN precipitation. You will find the protocol in Maniatis book.

Best

Olivier

There are another choices better than TRIzol nowadays. But if really need use this option, you can remove the excess of phenol adding more one step of cloroform extraction. After you add cloroform and separate the aqueous phase in a fresh tube, add an equal volume of cloroform, vortex 30 seconds and centrifuge 2 minutes 14.000rpm, transfer the aqueous to a fresh tube. Preciptate the RNA with isopropanol 100% and wash the pellet with ethanol 75% to remove salt and polysaccharids. Good look!

To get rid of contaminating phenol you can use 1/5 vol of chloroform and for any excess salts and even phenol-chloroform, have an extra step of 75% ethanol wash

washing twice with 70% ethanol which is to be prepared by using DEPC sol. can be done at the previous step before the final step.

Shortly:

Add 1/10 vol of NH4OAc, en 2.5 volume 100% cold EtOH (100%) and 1 µl glycogen (if you think you'll the pellet without see glycogen you can do without it)

mix

Incubate 30 min at -80°C

Centrifuge 20 min at 12000g at 4°C

(remove supernatant)

Wash with 75% ice cold EtOH

Centrifuge 5 min at 12000 g at 4°C

(remove SN)

Re-suspend the pellet in DEPC (can take some time)

we use it and it works.

PPT with NH4OAc is a good technique to remove salts. But before that a chloroform wash is also good. How do you lyse your sample for trizol extraction? A digestion with proteinase K, as said by Daniel C, is not bad too.

When we had the same issue (it is most likely related to remnants of phenol in your sample, what - sadly- happens often after TRIZOL extarction), an extra round of EtOH precipitation made a huge difference.

Ethanol precipitation will help.

try a purification of your RNA sample using phenol chloroform- Phenol should be water saturated

In my case, after the EtOH precipitation I use to dyalize the RNA sample against water or buffer. You can even do it by using one of those concentrating columns (VIVASPIN and so); in this case I use to perform several steps: 70% EtOH, H2O + EDTA (3x), H2O (5x)

After Trizol you can remove phenol using Chloroform. Transfer the aqueous phase to new tube then wash using Isopropyl alcohol. Next, wash RNA pellet with 75% ice cold EtOH. Dissolve RNA in warmed (55-60oC) RNase Free Water.

For ultra pure structural biology samples I dialyze RNA with a gradient of NaCl. Dialyze for at least 6 hours each in 1M, 0.5M, 0.2M and finally 0M NaCl buffer. This competes away all contaminants as proven by NMR spectra. You can get very small dialysis devices to deal with small volumes/concentrations.

I extract RNA from marine macroalgae, those tissues are rich in polyphenols and polysaccharides. Using CTAB extraction with little modification based on sample tissue gives a clean and clear RNA. Generally I use Chloroform extraction.

followed by Absolute ethanol precipitation and one more round of cl extraction follwowed by LiCl precipitation.

I also do RNA extraction using Trizol, and I was also getting low A260/230. A colleague from Research gate suggested wash RNA pellet two more times with 75% EtOH. It worked beautifully. Good luck ;-)

Ethanol (cold) /isopropanol (RT) precipitation 0.5 M salt (e.g. NaAc) is the best and commonest used method. As people suggested above...go for it!

When we used Trizol, as Kris suggested the problem was usually contaminating phenol, So we have added a chloroform extraction step prior to ethanol precipitation. We always follow with a 70 % ethanol wash to remove salt.

Low 260/230 ratios are frequently improved following a LiCl precipitation. Add 7.5 M LiCl (Ambion) to a final cc of 2.5 M (example: 10 uL LiCl 7.5M to 20 uL RNA sample, for a final volume of 30 uL). Follow Ambion protocol online. Alternatively, you can prepare LiCL 7.5 M yourself, and follow Ambion protocol.

It always work for me, with vegetable tissues. Good luck.

You could use chlorophorm extraction :-)

May be PD10 column can help since it allow you to get ride of low molecular weights components.

Hmm can't you just use column clean-up kits?

I use Megaclear kit (ambion) to clean up RNA samples. Alternatively ZR RNA miniprep (Zymo research) also does the job

Either do 2-3 additional washes with 75% Ethanol or simply use a commercial kit for better quality and higher yields.

I improved the RNA quality after 75% EtOH. But theses washes are ok for ribossomal total RNA, mRNA can be lost or degrade. Best

your low yield can be related not only to polysacharride, etc..., but to the extraction protocol itself. As suggested above, you may want to try the Prot. K to improve your yield. You may also want to try other method that Trizol. Qiagen or Promega Kit are good one, and you can get free samples. Also, time of extraction should be as short as possible as RNA degrade fast, but you probably know that. That is why the Kits are great for RNA, it take less than an hour to get your sample.

To prevent this in future:

I had similar problems with Trizol. By doing a centrifuging step at 12,000 G, 10 minutes, 4oC after homogenization and incubation of the trizol with the sample I was able to stop this. This is also described the in the trizol/trireagent procedure.

To fix the samples you currently have would expect it is best to re-extract them. Treat the RNA solutions as though they are your starting material. You can avoid any harsh homogenisation/ cell lysis steps though.

I don't worked on skin tissue but experienced extraction total RNA from fatty tissues (hippocamus of brain) using QIAzol Lysis reagent. some of critical points are: 1-incomplete homogenization, reduced RNA yield. 2- for samples containing high fat, proteins or extracellular material, centrifuge the homogenates at 12000 Xg for 10 min at 4oC to remove insoluble materials. 3- carefully transfer the upper aqueous phase to a new tube. according to my experience this stage is very sensitive and need to be more carefully. RNA isolation from fatty tissue is very difficult than other samples. but I could obtain good results because of worked very carefully especially in transfer stage.

I extract RNA from aphid heads. In my case, I realized it is important to remove all the supernatant after the centrifugation in chloroform:IPA (centrifuge, decant, spin, remove with pipette). I also do two ethanol washes. Good luck,

Q

Add 2 washes with 70% ethanol at the end. Decant and air dry for 5 minutes. Do not dry too much. Use 42 degrees warm elution buffer or NF water to resuspend

Yes, As Steve said you should give 2 washes in 75% ethanol which will get rid of all the contaminants and gives a clean prep. Your 260/230 will drastically improve. I did the same and it helped out.

All the best

i swear by these: http://www.zymoresearch.com/rna/direct-zol

best value on the market

The recommendations above (additional chloroform step, extra ethanol washes) are good, but the biggest purity improvement I found was when I started transferring the aqueous phase to an RNeasy mini column to complete the washes and elution. You loose some RNA in the column, so the your choice depends on the application and amount of starting material. My 260/280 improved from 1.8 to 2.1, 260/230 from 0.75 to 2.1, but yield fell from 30ug to 18ug.

I started with 60mg mouse skin, crushed with mortar and pestle in LN2, homogenized in 1.2ml TRIzol with TissueRuptor. pelleted debris 12000 x g 5' 4C, mixed supernatant with 1/5 volume chlorofom, phase separated in phase lock gel with additional 1/5 volume H2O (increases density difference between aqueous phase and gel to ensure good separation, but not always necessary). Removed aqueous phase and added 1/2 volume isopropanol relative to aqueous phase volume. I split the sample at this point to test manual pellet/wash vs. RNeasy mini. 

Manual: RT in isopropanol 10', pellet 12000 x g 5' 4C, twice wash 75% ice cold EtOH, dry completely, resuspend in H2O.

RNeasy mini: Transferred aqueous/isopropanol mixture to column, 8000 x g 15". Then follow the protocol: 700ul RW1 15", 500ul RPE 15", 500ul RPE 1'. To elute, incubate column in H2O for 5' (optional 55C to increase yield), 8000 x g 1' elution (optional re-elution with same eluate).

If your phase separation with phase lock gel isn't working well, you might try 1/3 volume chloroform and 1/3 volume H2O relative to TRIzol/homogenate volume.

Hi Nick Schaum,

I have been using the PureLink® Plant RNA Reagent from Ambion to extract RNA from difficult tissue (high in polysaccharides) to improve yield - Trizol and RNeasy mini kit didn't give much RNA.  The PureLink® Plant RNA Reagent  works wonderfully, got 1000x in RNA yield in comparison to RNeasy kit.  Now, I use the mini columns and the buffers RW1 and RPE for washing and I noticed that something from the PureLink® Plant RNA Reagent is interfering with RNA probes hybridization.  I am trying to enrich for my viral RNA from the total RNA by using specific designed viral probes.  This procedure works well when I use total RNA from a different extraction method but it does work on PureLink® Plant RNA Reagent RNA.  I believe that 2-Mercaptoethanol isn't being washed efficiently from the column, PureLink® Plant RNA Reagent has 20% of 2-Mercaptoethanol.  Have you experienced this type of problem before?  I see that you have modified the Trizol protocol to use columns as well.  Any help is appreciated.

Thanks,

WLD

Hi

I had a same issue with my samples. and interestingly after reading so many research gate answers, i found this paper http://www.ncbi.nlm.nih.gov/pubmed/19146820, using butonal at the end steps, which have shown improved the quality of the RNA/(260/230)/RIN etc..esp for low amount RNAs samples, interesting!!!!. Have to try

 Hi, i already isolated RNA from cultured cells from miRVANA isolation kit and i am getting good yield of RNA and 260/280 but 260/230 value is below 1.0.

Now can anyone help how i can now improve my 260/230 value or what to do to improve 260/230 value in near future using RNA isolation kit.

Please help

Thanks in advance

Rashi

Try to add amylase to digest the CHO and then pass your sample through PD10 column to get ride of the salts,may be this will help

I strongly suggest to follow Mojca Strazisar's suggesstion. I have used similar protocol when samples 260/230 =

Purify RNA with the Direct-Zol RNA minipreparation kit, follow their protocol and then wash once with 80% ethanol, I could get RNA from 50,000 cells with both 260/230 and 260/280 more than 2.0. Good luck.

Good suggestions above. Another simple method I found successful is:

Precipitate with isopropanol, centrifuge and wash with 70% EtOH, redissolve in RNase free water.

Assumption: you had dissolved RNA in RNase free water and stored it in -80 degrees - of course after getting upset with the low 260/230 ratio - may be the previous day.

1. Add ~500ml isopropanol to your the -80 frozen RNA sample

2. Incubate at -20 for 1 hour

3. Mix by inverting tube; spin at ~15000g for 10min; carefully discard supernatant

4. Wash pellets in ~ 750ml EtOH three times; air dry (with tubes facing diagonally downward on clean tissue paper)

5. Dissolve in RNase free water

6. Smile at the sight of the highly improved 260/230 ratio

Thank you Henry Awika! I had the same problem of Lucas and I used the protocol you suggested! All the 260/230 ratio are happily above 2, whereas yesterday some of them were under 1. Thanks again!

Henry Awika's method also helped me improve 260/230. Thank you!

Thank you Henry Awika for this suggestion. Just a quick question. I read that at cold temperatures isopropanol precipitates salts as well. Therefore, why incubating it at -20° C ?

Sara Blocquiaux I recommend reading this paper from Zeugin et al. from 1985: "Ethanol precipitation of DNA", which I attached to the answer. It shows that temperature has only a very minimal effect on the precipitation efficiency.

Christian Praetorius Thank you for the quick answer. Your paper however uses 'Ethanol precipitation' which is not the same as isopropanol precipitation.

Read:

https://bitesizebio.com/2839/dna-precipitation-ethanol-vs-isopropanol/

https://www.researchgate.net/post/Whats_the_difference_between_Ethanol_and_Isopropanol_in_nucleic_acids_precipitation

@Sara_Blocquiaux I know. But the general behaviour is the same. Isopropanol is less polar, which explains much of the difference. I would only use it when you have a big volume from which you want to precipitate, since Isopropanol is much harder to get rid off afterwards. It dries much slower.

Sara Blocquiaux Yeah it true when u precipitate the RNA from chloroform. The cold temperature brings up other contaminants as well. When we incubated in ice, most samples showed less 500 ng/ul) we usually get high > 1.8 (260/280). Since RNA conc is higher than contaminants in this sample it won't give much trouble. But if you're trying to isolate RNA from small volume sample (< 1-2 x 10^5 cells) it will give problem.

I strongly suggest incubating in room temperature.

If I m right. Henry Awika suggests to re precipitant the isolated RNA (dissolved in water) and then repeat the last few steps. This I have never tried. Better try one or two trial samples and then proceed to your main. ATB.

Karthikeyan Rajagopal

Thank you for the advice. Do you think 1h incubation at RT is necessary? We usually do 10 min at RT for precipitation with isopropanol.

Sara Blocquiaux

In my personal experience, I don't think 1 hour incubation with isopropanol will work. It will precipitate extra salts. When i have lesser volume than i go with longer incubation time but never more than 20 minutes. more than 20 minutes always precipitates salts.

Avneesh Kumar I agree. How much isopropanol would you add to the RNA dissolved in 25µl RNAsefree water?

Sara Blocquiaux Please follow Mojca Strazisar (Top recommended suggestion). It works 100% .

I usually isolate RNA from cells cultured on scaffold which always give high salt contamination and less yield. I tried above said method and it worked for more than 25 samples that had < 0.5 (260/280) and RNA yield less than < 200ng ( in total volume).

Only change is incubated overnight in -80 C instead of 30 minutes.

Reg Isopropanol precipitation method, use one volume of isopropanal and try to use glycoblue (1 ul (10-15 ug/ul), which help aggregate the RNA and the chance of losing during this process will be less.

( Reference: https://www.thermofisher.com/in/en/home/references/ambion-tech-support/nuclease-enzymes/general-articles/working-with-rna.html.)

This reference is for normal RNA ( > 1.8, 260/280). if 260/280 is less than

@sara blocquiaux

i always use 6 parts volume of isopraponol ( 600 ul in 1000 ul).

Mojcat Strazisar method: i had tried this one, but for me, addition of NH4Ac (a salt) is just increasing s alt concentration in your sample. I found better result with lithium chloride than NH4Ac.

Everything is depend on your sample's chemistry, so understand that and then try to modify protocol.

Sarah Blocquiaux, on the concern with salt precipitation at low temp, I believe Christian Praetorius and others have addressed it well. On my part, I haven't experienced a great deal of a problem resulting from the re-precipitation procedure I outlined.

On the question of how much isopropanol to add, I would comfortably say at least 75% vol; better equal to, or more than the RNA 'solution'. And by 'more than' I literally mean 'more', i.e., any amount as will reasonably fit the tube you are using, allow you to get a good spin-down, and be able t 'see' and recover your RNA. I have had success with literally 'punishing' stubborn samples with floods of up to 3X vol. But be sure you save your skin by noting the volume of the 'flood', just in case you will need to report this.

Thanks very much for useful comments and recommendations.

In our lab I use molecular grade glycogen to clean off salts and other contaminants that absorb at 230, and it performs wonderfully.

But I am not sure if the method can have any impact in RNA integrity.

I will appreciate to get a response from anyone with experience.