Hi,

TriZol is a phenol based chemical. Phenol carryover during RNA/DNA extraction significantly affects the isolated nucleic acid quality. Please check your spectrophotometers graph around 270nm if phenol "shoulder" exists. Phenol UV spectrum is about 270nm. So if your A260/230 is lower than 1.8, you must consider phenol contamination.

There some commercial RNA extraction kits work with phenol based lysis buffers (i.e. TriZol). They guarantee no phenol contamination.

Good luck

If you are using the trizol method, so better to change the tube when you are treating your sample with chloroform and try to dry the ethanol as much as possible in the last step, or after getting the RNA with the trizole method you can, try with commercially available cleanup kit specific for RNA.

Hi, how do you homogenize the 15mg of brain? Did you use the trizol protocol or

did you use a kit after the chloroform step? You wrote " after isolation and purification by kit " so I am confused.

Valeria

You can use RNA clean-up and concentration kit, I used NORGEN kit (https://norgenbiotek.com/sites/default/files/resources/RNA-Cleanup-Insert-PI23600-13.pdf)

I followed Protocol (B) for RNA Clean-up and Concentration from Phenol/GuanidineBased RNA (Trizol or Tri Reagent) Isolation Methods

its gave me a good RNA quality and quantity

good luck

Valeria De Arcangelis I have used 15mg of tissue and used a sonicator for homogenization. I haven't used any kit in the whole process of isolation. Purification was done using 3M sodium acetate.

Hi, sonication it is surely a good method for RNA extraction but it is also possible to shear the RNA. Maybe you have to pay attention to some conditions as temperature or change others?

You can try with another type of homogenization only to understand if you had problem with sonication or other steps.

I don't known your protocol steps so my suggestions are pointless because you already did it, but you can try with a ctrl (mouse?) brain, you can split in two the brain and try different homogenize step with the same extraction

You have to be sure of correct disruption of the sample and then procede with extraction. You can try to cut with sterile scissor the brain in trizol and then sonicate at 4°C. Or before putting the frozen brain in trizol you put it on dry ice, hit quickly with a small "hammer" (or something similar) and then put immediately in trizol to homogenize.

I suppose that you centrifuge after sonication, you can vortex after adding clorophorm, add sterile glycogen together with isopropanol and precipitate. Read the RNA see the results and then procede with 3M sodium acetate.

Valeria

There is no need in sonication when you homogenize 15 mg of brain tissue: TriZol contains guanidine, which is a very good lysing agent. Try to homogenize your sample with only pipette tips. When we isolate RNA with TriZol in such manner we always get about 1 mkg of total RNA from 10 mg of tissue.