I am facing problem in isolation of protein/RNA from newly generated organoid/spheroids embeded in matrigel. I know about the use of matrigel recovery solution (BD); however, looking forward to hear any alternative strategy.
The scond one is immunofluorescence analysis. Need suggestions beside OCT and parafin method, anyone have gone through direct staining without making sections ?
With Thanks
Imran Ullah I am not sure why you are having problems with RNA isolation.
You should be able to easily break up the matrigel and get your organoids out.
Just carefully remove the media from the wells, add ice cold PBS and gently pipette up and down until the matrigel is broken into tiny pieces. Transfer into a chilled centrifuge tube that has ice cold PBS and spinning down at 300g for 5 min at 4C.
Remember that matrigel will solidify at room temperature.
Once you have the organoids pelleted you can homogenize and proceed with basic trizol RNA isolation.
if you want, you can embed the pellet in OCT and snap freeze for later immunofluorescence analysis. OCT embedded tissue is perfectly fine for isolating good quality RNA as well.
hope this helps.
good luck!
I always harvest spheroids from matrigel using matrigel recovery solution (BD) for RNA isolation and WCL, but I wash the spheroids extensively with ice-cold PBS to remove matrigel traces before sample processing.
We routinely perform immunofluorescence in matrigel embedded spheroids with very good results. Follow usual IF protocol but after secondary antibody incubation include a high salt washing step.
Good Luck
To isolate RNA, use RLT buffer. Pipet 250 micro litres into single well of a 6 well plate, incubate for 3-5 minutes, transfer into eppendorf and store at -20 prior to processing.
To image, break up Matrigel with ice-cold PBS without too much pipetting as this will disturb organoid structure. Transfer organoids in PBS into an eppendorf. Allow 15-20 minutes for organoids to settle into bottom of eppendorf. Add permeabilising solution. Incubate. Allow to settle again...etc.
Stain as you normally would. Permeabilise-wash-block-wash-primary antibody-wash-secondary-wash. Final step; aspirate organoids in PBS and put them into a glass bottom 96 well plate. Image on confocal. This method requires no sectioning and/or embedding.
Good tip Rahul Kumar . We also recently made a switch to using Trevigen's Cultrex® Organoid Harvesting Solution. It works well
Good luck!
Ana Klisuric could you please tell, should RLT be added after disrupting matrigel bubble or directly to the well for future RNA isolation?
Many thanks in advance.
Afshan Iqbal directly to the well, it'll dissolve the materiel upon contact.
remove media first.
Ana Klisuric Rahul Kumar
Do Matrigel leftovers disrupt the Trizol extraction?
Maayan Pour I did with RNeasy microkit. Got high concentration and good quality. No interference with matrigel leftovers.
Maayan Pour No. We performed RNA-Seq experiments along with qRT-PCR without any problem
I am also having problems isolating RNA from mouse intestinal organoids. I am using the RLT buffer to break the matrigel dome and harvest it. I am using the spin column-based protocol. But the 260/230 ratio is around 0.7, and the concentration is around 45 ng/ul. Although the 260/280 is acceptable, 2.19 and good integrity as I can see proper 28S and 18S bands in the gel. Any suggestions are appreciated on how to increase the quantity of RNA.
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