Transcription profiling of 8-day-old Podocin-Cre, Z/EG double transgenic mice glomerulus and brain

ABSTRACT: Mouse glomeruli and brain capillary fragments were prepared. Podocytes were separated from isolated glomeruli from 8-day-old Podocin-Cre, Z/EG double transgenic mice as follows: Isolated glomeruli were incubated with trypsin solution containing 0.2 % trypsin-EDTA (Sigma-Aldrich), 100ug/ml Heparin and 100U/ml DNase I in PBS for 25 min at 37 degree, with mixing by pipetting every 5 min. The trypsin was inactivated with soybeans trypsin inhibitor (Sigma-Aldrich) and the cell suspension sieved through a 30 um pore size filter (BD bioscience, Franklin Lakes, NJ). Cells were collected by centrifugation at 200 x g for 5 min at 4 degree and resuspended in 1ml PBS supplemented with 0.1 % BSA. To separate GFP-expressing (GFP+) and GFP-negative (GFP-) cells, glomerular cell were sorted using a FACSVantage SE (BD, San Jose, CA, USA) operating at a sheath pressure of 22 psi. Autofluorescent cells were excluded by analyzing the emission of orange light (585 nm). To allow for standardization of results, the samples such as glomerular RNA, brain capillary RNA, rest of kidnay RNA, podocyte RNA and foxc2 glomreular RNA were hybridized in competition with common reference samples.

INSTRUMENT(S): Axon- GenePix4000B

ORGANISM(S): Mus musculus

SUBMITTER: Christer Betsholtz 

PROVIDER: E-MEXP-492 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress