Low A260/230 ratios are usually caused by phenol or salt contamination. Since you said that you do two chloroform extractions, it's unlikely to be phenol. However, precipitating at -20C will also precipitate salts, so that's likely the issue.

If you're starting with high cell numbers (>500k) then precipitating at room temperature will be fine. For lower cell numbers, you can precipitate at 4C with rotation for one hour. I never precipitate at lower than 4C because of the issue of salt contamination.

Ciaran Daly Thank you so much for your answer, but our main problem is, that after purifying with amonium acetate the A260/A280 lowers under 1.6 and nanodrop finds protein contamination, although before purifying there wasn't any contamination..

My tips would be

1) one chloroform extraction only (this is primarily to remove phenol carryover, so is 'optional' rather than 'essential'. Doing it twice is overkill.

2) room temp isoprop precipitation, for 20mins only. NEVER use isopropanol precipitation cold, because cold isopropanol will 100% precipitate guanidium salts. Cold isopropanol for 1 hour will precipitate a LOT of guanidium salts.

Note that ethanol will precipitate far fewer salts, but also precipitates nucleic acids more poorly (so needs cold temps)

Here's what I usually do:

• Cells + trizol, add chloroform for phase separation, mix well, centrifuge
• Collect aqueous phase (avoid interphase!), mix with chloroform (1:1 vol:vol), mix well, centrifuge
• collect aqueous phase, add glycogen (10ug) + 0.1 vols sodium acetate (3M, pH 5.5), mix, then add 1 vol room temp isopropanol. Mix well, incubate at room temp for 20 mins (sodium acetate is probably not needed, but extra +ve charges don't hurt).
• centrifuge in chilled centrifuge, max speed, 15 mins: you should see a pellet here, even if just from glycogen. Remove supernatant, discard
• wash (gently) with 75% ice-cold ethanol, spin at max in chilled centrifuge
• remove supernatant (pellet should slowly become more opaque and 'crunchier'), repeat this wash step
• remove all supernatant, air dry pellet at room temp in flow hood for ~15min
• dissolve in appropriate amount of nuclease free water, nanodrop -use 15-20ul as a first pass volume: you can always dilute more if it's too concentrated, but it's harder to concentrate up if it's too dilute.

Regarding QC: 260/280 is protein contamination: this will almost never be problematic, because the trizol protocol carries over almost no protein if you avoid the buffy interphase, so anything 1.8+ is totally fine.

260/230 is the critical ratio: guanidium salts are strong absorbers at 230, and all are very bad for downstream biochem. Aim for a 260/230 of at least 1.7, but ideally 2.0.

If this is bad, simply reprecipitate: add water to make the volume something manageable (like 500ul), add 0.1 vols NaAc (3M, ph 5.5), add 10ug glycogen, mix, then add 1vol isopropanol, mix, incubate 20mins at room temp, etc (see above).

Note, however, that it's a ratio, so very low [RNA] will give a poor 260/230 not because of salt contamination, but because of low [RNA]: for samples with RNA yields below ~200ng.ul-1, you can usually accept lower ratios.