When you say "bad", how bad? What sort of RNA yields are you getting? (low RNA yields inherently tend to give worse values, as the values are ratiometric: less RNA, same salt = lower 260/230)

How many cryosections are you using, how are you handling them, and how much Trizol are you using, per prep?

If you have decent RNA yields, you can always reprecipitate your RNA to clean it up: I routinely do this for any samples with 260/230 values lower than 1.7, and while you'll lose ~25-30% of your RNA, the remaining sample will be much cleaner (260/230 of 2.0-2.1).

I agree that if the 260/280 ratio of your RNA sample is off, just try to precipitate it and here is a previous answer for that in researchgate...

https://www.researchgate.net/post/Can_low_260_230_ratio_in_RNA_extraction_interfere_with_qPCR_or_RT-PCR_result

I have done it before it works well...

Good luck ,

Ali

Hi Chiara,

With my Trizol using experience, 260/230-260/280 ratio can be bad for the following reasons-

-Due to residual phenol that remained during your RNA purification step.

-some contaminants might have been introduced during any of the RNA prep stages. Since, RNA is really very very sensitive, handling and maintaining a sensitive and RNase free condition is always half success.

To get rid of the above, use no greater than 1ml Trizol and if you are doing Phenol-Choloform-ethanol precipitation, try to repeat Choloform addition stage twice, thus you may get rid of any excess Phenol. Best wishes.

My 260/230 are always lower than 1, by using 600 µm of cryosection and NO MORE than 1 mL of TRIzol. I always use RNAse free water, clean pipettes and cottoned tips.

"No more than 1ml trizol" is actually a harmful stipulation rather than a benefit: the dangers in trizol RNA isolation are more likely to stem from NOT ENOUGH trizol, rather than too much. I often find (particularly with fatty tissues or tissue sections with significant cryoembed) that using more trizol helps markedly.

If you have poor 260/230 ratios, this is indicative of guandium isothiocyanate contamination: trizol extraction is a guanidium based protocol, so this is not uncommon.

Guanidium salts can be easily removed by reprecipitating your RNA:

Make your RNA up to 500ul with DEPC H2O (you don't need to do this, but it makes the subsequent steps easier, as all the volumes are more easily pipetted).

Add 0.1 vols 3M Sodium acetate, pH 5.5 (so for 500ul of RNA, add 50ul).

Add 10ug of glycogen (optional: increases RNA recovery)

Add 1 vol ROOM TEMP isopropanol (so 500ul, here). It must be room temperature: if it's cold, it will precipitate salt along with your RNA.

Leave these at RT for 20 mins, spin at full speed in a benchtop, 10-15mins (chilled benchtop for preference). Wash 2x with ice cold 75% ethanol, spinning between, then allow to dry for 15-20mins in a ventilated hood.

Redissolve in DEPC H2O. You should have 70-80% of your original RNA (you always lose some) but it should be much less salty.