Dear Safieh Zeinali ,

I have isolated RNA from iPSCs grown on Matrigel using Trizol and Qiazol without any issues in RNA quality or concentration.

Our protocol is based on the one from ThermoFischer (https://tools.thermofisher.com/content/sfs/manuals/trizol_reagent.pdf)

I wash the RNA twice with 75% ethanol(ISOLATE RNA-step 2 on page 2).

Hope this helps.

Dear Shambhabi Chatterjee ,

Many thanks for your reply and sharing your experience. I will give it a go.

Cheers,

Safieh

Hi Safieh

Can I know if the Trizol protocol worked for you?

I work with primary mammary cells grown on a Matrigel layer in a transwell system. I've been using NucleoSpin RNA plus kit and my RNA extraction has not been successful..

Hi Zahra,

I had a comparison between Trizol and RLT plus buffer and RLT plus buffer and its kit (RNeasy Plus Micro Kit) worked more efficiently than Trizol based approach. So on the last day of culture I did these steps: 1. Remove the media and wash monolayer with 200ul ice-cold DMEM or PBS gently. 2. Add 200 ul ice-cold DMEM or PBS to transwell to dislodge the layer by pipetting up and down. 3. Transfer the transwell content(cell layer) to 1.5ml eppendorf tube and incubate it on ice for 10 min . 4. Spin it down at 1500G for 3 min and 4C. 5. Check the cell pellet and remove the supernatant (containing matrigel) 6. Add 150ul RLT plus buffer, pipette it up/down several times and then vortex it for 30 second. 7. Leave on ice for RNA extraction or store at -20C untill RNA extraction.

I hope it helps. Good luck.

Safieh