Dear Richa,

The 260/230 ratio depends on the concentration of your samples. If you have a very low yield - e.g. 10 ng/µl - you will get a low value in this ratio. Just for the record - the absorption at this ratio tells you about contaminants that are present in your samples as phenols and carbohydrates absorb light in similar ranges.

Per definition the 260/280 ratio should equal 2.0 to count as "pure RNA". If it is lower you should digest the sample with DNAse. In our lab we always do a DNAse digest with our samples to make sure that no DNA traces are left. This is a very crucial point since you don´t want any residual sample DNA in your later PCR to be amplified. You only want the transcribed cDNA to amplify in your qPCR, right?

Once you get rid of the DNA traces in your RNA and you clean up your sample you have a fair chance to get rid of contaminants as well. When cleaning your sample with phenol/ethanol (either washing the pellet or using it for clean up) make sure all phenol/ethanol has evaporated before resuspending/eluting the RNA sample. You do this by leaving the tube open for some time, in addition you can sniff carefully for ethanol/phenol traces. This will greatly increase the purity and will yield in higher 260/230 ratios.

If you don´t get the required quality you should do a new RNA extraction from fresh samples.

Good luck.

I was taught to re-extract the sample again if the ratio is low. I have not experience similar situation when I use the RNAeasy kit. Maybe you can re-run it using the kit.

This is an interesting question. Sure, the purity of the nucleic acid is an important issue; especially in regard to determining the real concentration of nucleic acid in your sample and whether the sample is pure enough to give reliable and comparable results in downstream applications such as qRT-PCR.

First, there are some terms mixed up in the question already: purity is not the same as integrity/quality. The measurement you talk about just measures quantity and purity of nucleic acids. If you are interested in quality and integrity, you have to run a gel or determine the RIN value.

The answer of Susann is a little bit confusing. RNA and DNA can hardly be distinguished by the measurements. Even a DNAse digest will not improve the A260/230 ratio per se. It definitely will mess up the A260/280 ratio because of all the protein in the sample. What may help is the cleanup of the DNAse digest. High A230 values in relation to A260 seem to be mainly contaminations from the Qiazol or RLT buffer. I assume it comes from carry-over of a little bit of Qiazol or pipetting to the rim of the Qiagen column. Best not to get any sample on the rim of the column, bet not load more than the 700 microliters.

Does the contamination that causes elevated A260/230 values matter? According to Qiagen: probably not:

"The efficiency of downstream applications depends strongly on the purity of the RNA sample used.  Pure RNA should yield an A260/A230 ratio of around 2 or slightly above; however, there is no consensus on the acceptable lower limit of this ratio.  Possible candidates that can increase the A230 include “salt”, carbohydrates, peptides, and phenol (or aromatic compounds in general).  In our experience, the increased absorbance at 230 nm in RNA samples is almost always due to contamination with guanidine thiocyanate, present at very high concentrations in the lysis buffer or extraction reagent used in most RNA purification procedures.

Please find an article discussing the effect of low 260/230 ratios in RNA preparations on downstream applications on page 7 of QIAGEN Newsletter 15, March 2010.  In summary, we found that concentrations of guanidine thiocyanate of up to 100 mM in an RNA sample do not compromise the reliability of downstream applications."

(https://www.qiagen.com/us/resources/faq?id=c59936fb-4f1e-4191-9c16-ff083cb24574&lang=en)

What they are saying is that high contamination with guanidine thiocyanate (which they claim is the main culprit of the reduced A260/230 ratio) do not seem to affect downstream applications.

However, your concentration measurement might be messed up by it since the contaminants will also absorb at 260nm. 

An important aspect to this is also that low overall A260 values give probably unreliable A230 values. This is what Susann means with the 10ng/microliters; then all your values and ratios become shaky and every little bit of contamination becomes "overrepresented". That is why Qiagen recommends to be careful with ODs below 0.15, and when in doubt, do the measurements at neutral pH since the ratios seem to be pH dependent. 

How to improve the A260/230 ration. I assume the safest way is to re-purify as Ru-Yeng suggests. Purifying by precipitating the RNA with alcohols and basically washing off the guanidine thiocyanate may work according to Thao Tran and Mojca Strazisar here on researchgate.

Thao Tran wrote here in a related question:

"My bloody experience: Wash the RNA pellet with at least 3x 75% EtOH at RT. Importantly, when adding EtOH, remember to roll and invert the eppendorfs so that it can wash any salts binding to the tube wall. You can gently resuspend the pellet when washing by flicking the tube. Washing EtOH at RT can remove the remanining

salts more quickly than washing with chilled EtOH, because the chilled EtOH make the pellet more solid, not contacting the washing buffer well." see: RNA samples with low A260/280 and A260/230 ratios - ResearchGate. Available from: https://www.researchgate.net/post/RNA_samples_with_low_A260_280_and_A260_230_ratios [accessed Oct 21, 2015].

Mojca Strazisar wrote:

"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)". See: What´s the best way to remove salts, phenol and other contaminants from my RNA samples? - ResearchGate. Available from: https://www.researchgate.net/post/What_s_the_best_way_to_remove_salts_phenol_and_other_contaminants_from_my_RNA_samples [accessed Oct 21, 2015].

Thank you Thomas for this very concise and exhaustive answer!

 Thanks Susann! I always wondered what the A260/230 ratio really means and I often ignore it. The problem is, if you have 10 RNA preps and one has a poor A260/230 ratio, do you precipitate all the RNA samples again or just one? Since RNA is finicky, each and every extra step may introduce more "severe" contamination in form of RNAses. So, in the end I want to know how much RNA is "roughly" in my preps, and equally important, what is its quality/integrity. The nanodrop does not give you any indication about the second aspect. I feel now better myself having checked it out more thoroughly, since RNA extractions are a backbone or starting point for many of my downstream experiments.

Out of curiosity. What will the RNA be used for. Many methods such as cDNA preps for next generation sequencing are very tolerant of input RNA quality.

thank you everyone for your detailed replies. so I have two options

1. re-extract using a mini kit?

2. re-wash using ethanol.?

will this decrease the yield of RNA..?

Hi Richa,

you bet it will reduce the yield. Very few things in life are 100% and cleaning RNA and precipitating nucleic acids are not 100% efficient. You may not have to clean-up the preps at all. The one with the very low quantity may not be useful. I would rather omit that sample than clean all of them again. I probably would avoid samples #2 and #10. Why do you think is there such a big difference in concentration of RNA between your samples?

Hi Thomas,

I think the difference is because I used mini kit it would be better if I had used micro kit as these were new born kidneys. The ones which were cystic and bigger (#3,11,16) gave me better yield than the smaller other ones.. or  may be there's some other reason.

or may be because the other kidneys in RNA later were stored for a long time that made them hard for homogenising..

thanks for your detailed answer David..I have some genomic DNA contamination as well but my probes span exon junction so i guess it should not affect the q-PCR results..?

HI! Richa Sharma! how are you?

Finally Have you resolved your problem? I have the same trouble, but I thinks it is due to the collection process. Thanks. 

Hi Alicia,

I  extracted more RNA because after  re-purification the yield was low.

However , afterwards I had the same problem and  then I talked to Qiagen about the low 260/230 ratio and they informed me that as long as 260/280 ratio is around 2.00 I need not worry. Its good for q-PCR.

How did you collected your samples.. and how about your 260/280 ratio. Are you going to use it for qPCR or some other higher technique?

I have just read (I found this topic also here) that you shouldn't be caring much about low 260/230 ratio. The most important is that you really have your RNA and yes you do. I have previously isolated RNA from leukaemic blasts and we did not even check this ratio (just 260/280) and we were using it for two-step PCR. Worked fine (many people confirmed that here). I don't know what about 0.04 ratio ha ha, but give it a try first and if you have any problems go back to that and try to wash it again, but remember that washing always causes the material loss.

As a preface, i'd like to say that low 260/230 ratios are usually from 2 main sources, protein contamination and guanidium salt contamination, with protein contamination being the more serious issue (guanidium doesnt seems to affect downstream reactions -https://www.qiagen.com/us/resources/faq?id=c59936fb-4f1e-4191-9c16-ff083cb24574&lang=en). However, protein contamination can usually also contain RNAses that are refolded in the aqueous phase, and this might affect the levels of certain genes. (from my personal observations). They can be differentiated by looking at the Nanodrop absorbance spectrum, protein ususally shows a sharp peak at 230nm, while guanidium peaks around 225nm.

I usually improve my 260/230 ratios by doing a re-precipitation with sodium acetate / ethanol. If you get some precipitates or gunk, try to dissolve them as best as you can after adding the sodium acetate, then vigorously vortex again after adding ethanol (3x10s). Thereafter incubation at -20oC overnight. Next day is just pelleting, then 2x 70% ethanol washes and elution. I usually get 260/230 of 1.8-2.4 after this. Note: if your RNA concentration is

I work with tomatoes. there is any information about high quantity of polysaccharides?  I always get low proportion of 260/230. I am afraid that is phenol contamination, and then, will ruin my next steps: making cDNA and qPCR. Thanks in advance.

Hi Larissa, 

I work with mouse tissue so I don't have any information on how the polysaccharides can affect the  RNA quality or quantity.

With my limited knowledge I guess It will not be affected for qPCR  as long as the 260:280 ratio is 2.0. If you fear for contamination it will be better to redo the extraction because if you try to wash it afterwards you will be left with very small amounts of RNA. You can also contact the Kit manufacturer with your question. Please find few useful links to papers about RNA extraction from plant tissues with high quantities of metabolites. 

I hope this helps...

http://www.sciencedirect.com/science/article/pii/S0254629912001214

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3597135/

This is not what you want, but I think you should still be able to run a qPCR with this low value, if the endogenous control is comparable for a Ct value with other samples, then you are safe in my opinion.

Hello Thomas Andl and Richa Sharma I am also facing the same problem while extracting RNA from tissue biopsies, using normal Trizol method (Invitrogen), I get good 260/280 ratios but 260/230 are not good. I re-precipitated the RNA sample using Isopropanol and 75% Ethanol washes, I get good 260/230 rations, but when I run the same samples on MOPS gel, I do not see good integrity of RNA.

Can you please suggest and help me trouble shoot this, if re-precipitating with Isopropanol is degrading the RNA?

Should I be doing column purification using (Qiagen RNAeasy kit). I have already Isolated RNA of these tissue (for some of the patients), I don't want to lose these samples, It would be great if help me troubleshoot this. I want to send these samples for next generation RNA-Sequencing.

Hi, Sharma

I have the same problem with you. I had reasonable 260/280 ratio. But the 260/230 ratio is too low around 0.2 . Since this question is 4 years ago, I thought you have solve this problem. Could you tell me what is your solution?

Thank you very much!

Hey guys, please see my answer above. The low 260/230 ratio can be due to either protein or guanidium contamination. If the peak is at 225nm, it's guanidine, if 230, its protein. To get rid of guanidine, usually a sodium acetate ethanol precipitation is effective. If protein, it is possible to do a reextraction with phenol chloroform, but you will lose yield. I highly recommend the use of phaselock tubes in RNA extraction, which both increases yield as well as decrease possible protein contamination.

Hey David,

Thanks for the detailed reply and corrections. I appreciate the help. I was under the impression that peptide bonds could absorb at 230 nm, which gives rise to the 230 nm peak, whilst guanidium isothiocynate from trizol was the contaminant at 225 nm (as attached above).

I think we can agree that the 225 nm peak is likely from guanidium isothiocynate. The 230 nm peak is a bit less clear. Its not guanidium isothiocynate (as above), and it is also not phenol, because otherwise the 260/280 ratio would be affected. It is also likely something not easily soluble in either water or ethanol, as a NaAc/EtOH re-precipitation would remove it (which it doesnt). The presence of a 230 nm peak also causes downstream problems with both qRT-PCR and in vitro translation (in my hands), which leads me to think that it is some undissociated ribonucleoprotein complex. It could be carbohydrates, but glycogen, a common carbohydrate carrier used to precipitate RNA doesnt give problems at even 25 ug used per 50 ul sample.

So this is my rationale for my conclusions, and i would appreciate further input!

Many of the above suggestions is true, but please be careful not to make universal assumptions. 260/230 ratio will indicate contamination of different sources depending on the sample´s nature. For some it may indicate presence of carbohydrate, for others plant derived secondary metabolites, and probably for most phenol residues. Thus consider from which type of sample you are extracting the RNA and always keep an eye on how it looks your aqueous phase after chloroform extraction.

Try doing reprecipitation with 10M LiCl after dissolving your RNA sample. This really worked for my plant RNA samples. Richa Sharma

I have also kept my samples in RNA later for more than 2 years and the integrity of the RNA was not compromised. If you are working with trizol, you should not touch the protein phases, and also neither the phenol, otherwise you carrier these contaminants. I have always worked with RNA purity major to 1.8, the best 2.0, but I have noticed that some colleagues have worked with RNA purities from 1.4 and they said, it works also well. It depends if you need it for further assays, if it is the case, I would not work with purities less than 1.8

Good look!

You have to reduce the polysaccharide contamination.

Hello all,

I have problems in RNA extraction from throat swab samples using Gn-isothicyanate lysis buffer and GnHCl wash buffer. The ratio of OD260/280 gives a very low value and is confirmed by the internal control inhibition by 5-10 cycles when compared to Qiagen results for the same sample. Is the concentration of GnHCl in the wash buffer that I am using is high or do I have to add some more additives to it? The pH of the wash buffer is 6.6.

I am facing the same problem with a 260/230 ratio of about 0.80. What could be the possibility? I can't figure it out. Can anyone help me in this regard?

Can some suggest, method of RNA extraction from Nicotiana benthamiana plants? I am having issue of this 260/230 ratio below 2.0. I am using TRIzol kit to extract RNA.