Dear Rishabh Kala

As you know, absorption at 230 nm can be caused by contamination by phenolate ion, thiocyanates, and other organic compounds. For a pure RNA sample, the A230:260:280 should be around 1:2:1, and for a pure DNA sample, the A230:260:280 should be around 1:1.8:1. Moreover, the peak at 230 nm is due to absorbance by the peptide bonds of proteins while the 280 nm peak is due to absorbance by the rings of aromatic amino acids (tryptophan, tyrosine, and phenylalanine).

Please see:

UV Absorption pdf for more information.

All the best,

Abdolvahid

Hi Rishabh Kala

Following things you may consider for your extraction process

1. fully homogenize your sample

2. careful about transfer of supernantant, as phenol/chloroform contamination gives you bad A260:280 ratio

3. Use 99.5 or more concentration of ethanol for precipitation

4. Clean your working area to avoid contamination as much as you can

5. mixing of your chemicals should be appropriate, too poor or more shaking may degrade your RNA

Thanks

Rishabh, Qiagen has a guide with some insight on this matter, available at: https: //www.qiagen.com/us/resources/resourcedetail?id=19988eb0-6df5-4d26-8435-33efe98197da&lang=en

Given the chemistry of the QiagenRNEasy kit, the contaminating reagent is almost certainly quanidine thiocyanate in the RLT lysis buffer. This should be removed during the RW1 and RPE wash steps. Unfortunately, the format of Qiagen's columns permits a small volume of reagent to remain 'stuck' in the tubes, especially when using fixed-angle benchtop centrifuges. You can often see this if you check inside the column after a spin. This makes the washes somewhat inefficient. You might reduce the 230 nm absorbance by manually removing this carryover contamination (e.g. with a fine-point pipette), or by performing multiple RW1 or RPE washes.

Alternately, you may be able to reduce the 230 nm absorbance by performing a subsequent preciptiation - but that's a lot of work.

I'd also like to mention that it doesn't take much quanidine thiocyanate to cause high 230 nm absorbance. Trace amounts of quanidine thiocyanate may not actually cause serious issues for whatever your downstream application is. For instance, RT-qPCR is generally unaffected by high 230 nm absorbance.

Brian Bower Thanks for your reply. I will try to do a multiple wash. Problem is one of there wash buffer also has Guanidine thiocyanate as one component.