Make sure you eliminate protein and ethanol contamination, as these will impact the 260/230. If you nanospeck it and see a peak at 230, it is probably ethanol contamination. If you see a peak at 260, then it is probably protein contamination. It is important to only take the interphase during the trill-chloroform separation. I always leave just a little interphase to make sure I don't disturb the protein (pink stuff on the bottom. Make sure you completely dry the pellet before resuspension to eliminate any residual ethanol. I normally do this in a hood. I take the tube, open the top, place it in a cracked petri dish until I cannot see any more drops of ethanol. I hope that helps.

Hi Sepideh

My suggestion is treatment the plasma with 20 micro liter of proteinase K at 56 ° C for 1 hour before using trizol. This will decrease the plasma protein concentration and increase the 260/280 ratio.

thank you so much for your guiding. I will try it hope to achieve good result

Mahin Gholipour David Farringdon Spencer

hi Sepideh

I think your protocol needs some modification

1)400 ul plasma + 4 ul proteinase K (10 min incubation on ic) after 10 min add 1000 ul trizol in a same microtube and shake them

2) incubated 10 min at room temperature

3) add 200 ul of cold chloroform for 20 second shake

4) incubated 5 min at room temperature

5)microcentrifuge: 4 c, 15min, 12000

6) 600 microlitr from the supernatant phase take and put it to new microtube with (recommend) 600-800 ul cold Isopropanol for 5 second we shake it and incubated for 10 min at room temperature(it's better to incubate it overnight at -20)

7) microcentrifuge: 4c, 15min, 12000

8) depletion of microtube's fluid

9) add 1000 microlitr of ethanol(75% ethanol in DEPC treated H2O)

10) microcentrifuge : 4◦C, 5min, 7500

11) remove ethanol completely (do step 9,10,11 again)

12) dry for 5min at room temperature (until ethanol completely evaporate)

12)add 20 microlitr of DEPC treated water

Good luck!

David Farringdon Spencer

HI mahdieh

first thanks for your guiding but I have a question about proteinase k, I have it and it's concentration is 20mg/ml

as you said i should use 4 ul from it, is that right ??@ Mahdieh Safarzad

Dear Dr. Gholipour

You should notice that Trizole backbone contains Guanidine salts, therefore, if your washing with ethanol does not conduct well you may see a high absorbance at 230. I recommend twice washing with Ethanol 70/75% followed by a washing with Ethanol 96%. Guanidine salts disrupt proteins structures and there is no need for Proteinase. For improving absorbance at 260nm, after using phenol-Chloroform- Isoamyl alcohol you can separate RNA by additional step of adding Chloroform alone and centrifuge. Of course extending incubation time of the lysates with Isopropyl alcohol at 4 centigrade degree can help to improving RNA concentration.

yes,that's right@

Dear Alireza / Dear Sepideh

Based on my experience proteinase k is so helpful for RNA extraction from serum(plasma). Proteinase K is an enzyme that cleaves the peptide bond in proteins next to the carboxyl group of hydrophobic amino acid residues (aliphatic and aromatic). What this means in a practical sense is that Proteinase K will work much better in a chaotropic salt (guanidinium isothiocyanate ) and at elevated temperatures. It is often used at higher temperatures (50-65 degrees C). High temperatures, help to denature proteins, exposing many more hydrophobic amino acid residues that would normally be hidden in the hydrophobic core of the protein. How much Proteinase K you will want to use depends on what tissue type you are working with. I think 10-20 uL of 20mg/mL Proteinase K for 400 Microliter plasma lead to a good result.

Good Luck