Hi Nichole,

By the numbers it looks like it's most likely salts so I'd not bother with the chloroform step again. Then again do you really need the sample to be in tip-top shiny briny condition? Most enzymes would tolerate a bit of salt, especially after you dilute the sample in a larger volume of reaction mixture. Personally i'd just go forward. BUT

https://www.thermofisher.com/uk/en/home/references/ambion-tech-support/rna-isolation/general-articles/the-use-of-licl-precipitation-for-rna-purification.html

This is a good guide doing precipitation on RNA with several parameters tested. This article uses LiCl but ammonium acetate should work, despite being more prone to salt contamination in the downstream as described in the article. Personally I tend to keep the volume low, aiming for about 200ul after addition of alcohols, but this is probably a personal thing.

Ian Hu I do need them in tip top shape because they are going for Sequencing. I thought about LiCl, but I (and others in my lab) have had 50/50 results and have lost samples so I was not confident in that route to be honest —but only because we haven’t narrowed down the problem with our protocol there.

hi Nichole Welch

i found this answer highly recommended while searching for purity issues during RNA purification. i think you should try it on one or two samples to see if it works.i dont honestly remember where, but i saved it

.

Absorbance at 230 is usually guanidium (270 is phenol). As a chaotropic salt it's obviously not a good thing to have hanging around, and yes, it will definitely interfere with downstream cDNA synthesis.

Good, clean, RNA should have a 260/230 ratio greater than 2, but I've generally found that anything about about 1.7-1.8 is acceptable.

Some guanidium often co-precipitates with RNA (especially if you use isopropanol at low temperatures) but it can generally be removed by simply reprecipitating the RNA. I tend to use the following:

Make RNA up to 500ul with H2O (larger vols make everything easier)

Add 50ul of 3M NaAc, pH 5.5 (+ 10ug of glycogen if you want to maximise yield)

Add 500ul of room temperature isopropanol (room temp is important: cold isopropanol will just co-precipitate your guanidium again), mix well, leave at room temperature for 20mins.

Pellet RNA by spinning for 10 mins at 12-13k rpm in a benchtop (chilled to 4 degrees, ideally), and wash 2x with ice cold 70% ethanol (just gently pipette the ethanol down the side of the tube so you don't dislodge the pellet, use maybe 300-500ul, then spin, remove, repeat).

Air dry 15mins, resuspend in whatever volume you think is appropriate (tailor based on expected yield).

I use the above protocol on any samples with 260/230 ratios lower than 1.7, and while you probably lose about 30% of your RNA in the process, the remaining RNA is invariably of much better quality (260/230 > 2.0, suitable for cDNA synthesis).

The protocol mentioned by Ahmed Abdelhamid works ok, but you might lose a 50% of your RNA

Update: I ended up following a basic LiCl protocol, however, I kept the volume small (50ul)

20ul LiCl

24uL Sample (I had resuspended in 25uL, -1ul for Nanodrop)

6uL Nuclease Free H2O

Let sit in -80* for 1 HR, spin at 16000xg for 15 mins, wash 2x with 70% EtOH. Samples came out with 2-600ng/ul RNA, 260/280 >1.9, 260/230>2.1

Nichole Welch How much RNA have you lost from your original sample??

André Lasalle This is completely a guess but it looks like only about 20%. Many of the samples read at a higher concentration after cleanup than previous and I presume that is because the high salt concentration messed with the curve therefore effecting the numerical readout. Some of the samples that Initially read at around 600ng/ul are now reading between 425-590ng/ul.

Nichole Welch excelent cleanup!!!! thanks for your answer