Optimal RNA prep from whole mouse tissue

Noa · 03-14-2019, 01:14 AM

I have an experiment planned in which RNA samples will be collected from total mouse tissue (eg whole kidney, whole brain, etc).
I plan to use Qiagen RNeasy kit for RNA prep and then do downstream library prep and RNASeq.

Since I want to get data on whole tissue RNA content (and I do realize I will only see very large-scale changes since any change occurring in a subset of cells will likely be invisible to me here) I would like to homogenize the entire tissue (and not just a portion). I will need to collect the entire tissue at the end of the in vivo part of the experiment and later process for RNA

What would be your recommendation on tissue preservation (given the size of a whole mouse organ)? What would be the best way to perform initial homogenization of the entire tissue? After this I can subsample a portion of the total homogenized sample for downstream RNA preparation and sequencing

Thanks
Noa

Marc_Jones · 03-14-2019, 02:43 AM

I would take 5mm x 5mm blocks of tissue and snap freeze them.
Macrodissect and homogenise in RLT using a hand homogeniser, or use a bead mill homogeniser.

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03-14-2019, 01:14 AM	#1
Noa Member Location: haifa israel Join Date: Jun 2011 Posts: 62	Optimal RNA prep from whole mouse tissue I have an experiment planned in which RNA samples will be collected from total mouse tissue (eg whole kidney, whole brain, etc). I plan to use Qiagen RNeasy kit for RNA prep and then do downstream library prep and RNASeq. Since I want to get data on whole tissue RNA content (and I do realize I will only see very large-scale changes since any change occurring in a subset of cells will likely be invisible to me here) I would like to homogenize the entire tissue (and not just a portion). I will need to collect the entire tissue at the end of the in vivo part of the experiment and later process for RNA What would be your recommendation on tissue preservation (given the size of a whole mouse organ)? What would be the best way to perform initial homogenization of the entire tissue? After this I can subsample a portion of the total homogenized sample for downstream RNA preparation and sequencing Thanks Noa

03-14-2019, 02:43 AM	#2
Marc_Jones Member Location: UK Join Date: Jun 2017 Posts: 10	I would take 5mm x 5mm blocks of tissue and snap freeze them. Macrodissect and homogenise in RLT using a hand homogeniser, or use a bead mill homogeniser. Last edited by Marc_Jones; 03-14-2019 at 02:45 AM. Reason: Clarity