Not necessarily. Various factors lead to bad sequence quality so you cant pin it down to just the 260/230 ratio and at least your 260/280 ratio is acceptable. Your PCR product purification reagents can also have an effect on this and even your extraction reagents. Getting a high concentration does not necessarily equate to a good sequence as well, I have had really bad quality sequences coming out of good concentrations. I hope the attached materials assist you further.

*Usually adding the washing step during PCR product purification (gel extraction or not) helps in removing impurities that affect the absorbance.

http://www.lsbfg.uwa.edu.au/procedures/causes-of-poor-sequencing-and-failures

The 260/230 ratio less than 1 shows more contamination of proteins and carbohydrate in your RNA samples. However, it would be better if you perform quantification of RNA on Bioanalyser to analyse the RIN number (RNA Integrity Number) or you can also check the quality using MOPS gel. If the RIN number is greater than 8 than you have a good quality of RNA and you can proceed to the cDNA library preparation for sequencing.

If you are using the illumina platform for sequencing, then during the cDNA library preparation (using TruSeq RNA Library preparation kit), the bead purification method is performed in its initial steps to remove the impurities such as ribosomal RNA, Proteins and other carbohydrates, hence only purified mRNA was found to be bound to RNA purification beads discarding other impurities. The higher impurities may lead to inefficient binding of the mRNA to Beads and to overcome this hurdle you should use 4 µg of RNA for library preparation.

What you see is mostly polyphenols and carbohydrates at 260/230. Some of them may be polymerase inhibitors, and that is where you may want to take a closer look.

If you have enough sample, try performing a SPUD assay and comparing the Cq to detect any significant change between a clean control and your samples.

http://www.sciencedirect.com/science/article/pii/S0003269706000856

Hi Angela,your question is very interesting and the answers provided here are really informative.

I have extracted RNA and DNA for NGS, but my experience is small. Therefore, I do not have a specific answer but I try to contribute to this topic wtih some general comments

If your samples contain some suspended material the spectrophotometric assays can be biased and sensitive to dilution. If you have enough RNA and the appropriate instrument, I suggest to repeat the assay with replicated RNA dilutions in a standard spectrophotometer.

Some tissues are very rich in polysaccharides and it is very difficult to reach the optimal RNA or DNA purity. We do not know what organism you analyse. However, the 260/230 ratio of your samples is very low. In RNAseq a good RNA integrity (measured by RIN) is more important than RNA purity (see Romit's answer). May be your samples work well in RNAseq, but the very low 260/230 ratio suggests that your purification protocol needs some optimization.

To get a proper solutions to your specific case, and possibly improve the quality of your samples, you should specify the organism and tissue that you analyse and the protocol for RNA purification

For a fast evalution of RNA integrity and genomic DNA contamination, you can simply run 100 - 500 ng of RNA in a standard 1% agarose gel, 0.5X TBE buffer at 4-5 V/cm. Although this is not a canonical MOPS denaturating gel, you will able to distinguish the 18S and 28S bands and any smear of degraded RNA.

I wish you a good work

Hi Angela,

The value you got of 0.68 from the ratio of 260/230 shows that your extraction protocol didn't clear away the unwanted proteins/carbohydrates/phenol or any other contaminants that which absorb at 230nm. The rule of thumb is that, the higher the 260/230 ratio, the cleaner, purer your sample is. The general values should range between 2.0 - 2.2. Kindly note that the ratio 260/230 of 'pure' nucleic acids are relatively higher than the respective 260/280 values.

For you, I'll suggest two things; run your RNA sample through a MOPS denaturing gel and run them through a bioanalyser, if you have. The current practice nowadays is to determine the RIN before doing any RNA seq

Hello Angela,

Could you solved your problem? How it went by sequencing with that low ratio? I'm in the same situation right now.

Hi Angela,

I'm also wondering how your sequencing went. I have the same problem, A280/A230 ratios between 0.15 - 0.95 but I am told this ratio is less reliable as a contamination indicator when your RNA qty is low, which mine is, between 21.8 - 93.1 ng/ul.

I'm also using Nanodrop (because we are waiting on a Qubit delivery) and my samples are whole aphids, which are darn tiny and hard to crush. I also wonder if their high sugar content could be adding to the ratio problem since some have mentioned a polysaccharide absorbance at A230.

Also, I just wanted to say thanks to all the people who provided great answers and insight, all these have been really helpful :-)

Angela Salzano Michelle Mak Mariana Batista Santos

how did you get the final RNAseq results. I use RNeasy Plus mini kit, have the same problem of low 260/230 value. the values are as low as 0.2 to 0.9.

should I go with RNAseq or should I go with RNA clean up process.

Hey everyone, how were your results? Did you go with RNA seq at a low 260/230 ratio? Harish Sudarsanam Michelle Mak Angela Salzano

Napat Emdee I had read and even talked to the Qiagen representative and it seems that it shouldn't be a big deal. but then I was also able to resolve this problem by doing multiple washes with incubation for about 5mins in wash solution before spinning it down.

Harish Sudarsanam Thank you for your information. Actually, I've already sent my low 260/230 RNA for sequencing. Lucky me, it seems there is no problem. I get the sequence results today. : )

Hi,

I am also getting really low A260/230 (>1.0) values and have to send my samples for miRNA-sequencing. Did the low A260/230 values influence the sequencing? My RIN values are all >8 (which is great) and my concentration values are between 39.4 - 276.44. My A260/280 score range between 1.34 - 3.45 (mostly meeting the criteria of 1.8-2.2). I really have to send my samples for sequencing ASAP but my supervisor and I aren't sure about the low A260/230 scores, we don't know if we should maybe consider a clean-up kit ?

Harish Sudarsanam I have been having the same problem with Qiagen. How did you go about the multiple washes to solve this problem? Thanks!

Danielle Jansen van Rensburg Angela Salzano apologies for delayed reply but probably it hepls others in future. I did have similar problems, Qiagen says that this has been a problem for a long time but it is also mentioned that it does not really affect the sequencing as there are numerous washing steps during library prep. But just for my satisfaction, I performed additional washing steps, it only takes extra 10mins and some more wash buffer but you are really sure now. Also, this does not affect the RIN value, all my RIN values were more than good. attached is the protocol.

is it possible to do DNA-seq analysis with bad 260/230 ratios?

hi

I am getting the low value, sometimes 260/230

My 260/230 ratio is 1.8 can I use it for RNA Seq.