Optimization of RNA extraction from laser captured microdissected glomeruli from formalin-fixed paraffin-embedded mouse kidney samples for Nanostring analysis.
2019
Optimized protocols for the microdissection
of specific areas from archival tissues and the
subsequent RNA analysis are needed but challenging
due to RNA degradation and chemical modifications.
The aim of this study was to present the most
appropriate protocol for utilizing mouse FFPE kidney
for laser capture microdissection and Nanostring gene
expression analysis.
We evaluated different section thicknesses (3, 5, 10
µm), 2 RNA extraction kits (Qiagen and Roche) and
different H&E staining methods to optimize
microdissection and RNA extraction from glomeruli and
cortical tubules samples from FFPE mouse kidney. RNA
quality and quantity were assessed via Nanodrop and
Qubit.
The protocol providing the best results consisted of 5
µm sections, a shorter protocol for H&E staining, and
RNA extracted with the Roche kit. Higher Nanostring
gene counts and lower qPCR cT significantly correlated
with RNA concentrations measured with the Qubit, but
not with measures obtained with the Nanodrop. The
Nanostring data showed that none of the genes included
in the panel was differentially expressed in the cortical
tubule compartment compared to the whole kidney.
However, 25 genes were differentially expressed in the
glomerular compartment compared to the whole kidney.
Our data showed that sufficient RNA can be extracted
from small compartments like mouse renal glomeruli
from archival FFPE tissue, and that whole kidney
analysis does not accurately represent the transcriptome
state of the glomeruli, which comprise only a small
proportion of the overall kidney volume.
Keywords:
- Correction
- Source
- Cite
- Save
- Machine Reading By IdeaReader
38
References
2
Citations
NaN
KQI