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.

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.