Hi everyone,
I’ve been trying to extract RNA for RNA-seq from iPSC-derived motor neurons, but unfortunately, the RNA quality has been too low for sequencing (RIN values around 5–6).
My current protocol involves gently scraping the cells, centrifuging to obtain a pellet (5 min 500g), then using Trizol followed by chloroform separation, and finishing the extraction with Qiagen’s RNeasy Mini Kit. I’ve been thinking of potential improvements — apart from trying trypsin and increasing the cell number, I’m a bit short on ideas.
Has anyone successfully extracted high-quality RNA from iPSC-derived neurons? I’d really appreciate any tips, tricks, or key steps that worked well for you!
Thanks a lot in advance!
To increase RNA yield, you need to flash-freeze the cells in liquid nitrogen. Do not let them thaw until you add the first buffer from the kit.
The Qiagen kit has specific steps for how to use cultured cells (max loading volume, any additional wash steps, etc).
As an alternative to Katie A S Burnette 's idea, you could also just "add trizol directly to the cells, THEN scrape them off". Trizol pretty much unfolds/denatures/dissolves everything, so RNA in trizol is protected from degradation.
So: plate of cells. Remove media, wash 1x with PBS (optional), add trizol, scrape with cell scraper. Collect the gloopy, stringy pink mess this will probably result in, pipette mix and/or vortex it to homogeneity, extract RNA.
If in doubt, use more trizol than you think you need. I used to use ~200ul for cells in 6-well plates, but for flasks I'd use at least 1ml.
Also worth noting: most RNAseq pipelines today will work just fine with a RIN of 6. RIN of 5-6 means "this isn't great RNA, but there is still intact RNA here". Probably if you submitted your samples, you'd get usable data.
It's only when you get down to RIN 3 that you're looking at "this is just degradation smears"
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