Method for analyzing RNA following intracellular sorting (MARIS) Of nephron progenitors (Version 1.0)

Version

1.0

Notice

This page is the corresponding protocol tomestone page generated as part of the ATLAS-D2K shutdown in July 2025. Many links on this page may be broken.

Authors

Andrew McMahon; Albert Kim; Riana Parvez

Keywords

[‘maris’, ‘kidney development’, ‘Intracellular staining’, ‘FACS’, ‘RNA-seq’, ‘Six2’, ‘nephron progenitor’]

Subjects

[‘Isolation, purification and separation’, ‘Immunological techniques’, ‘Gene expression analysis’]

Release Date

2016-08-01

Abstract

Transcriptional profiling is a key technique in the study of cell biology that is limited by the availability of reagents to uniquely identify specific cell types and isolate high quality RNA from them. We report a Method for Analyzing RNA following Intracellular Sorting (MARIS) that generates high quality RNA for transcriptome profiling following cellular fixation, intracellular immunofluorescent staining and FACS. MARIS can therefore be used to isolate high quality RNA from many otherwise inaccessible cell types simply based on immunofluorescent tagging of unique intracellular proteins. As proof of principle, we isolate RNA from sorted human embryonic stem cell-derived insulin-expressing cells as well as adult human β cells. MARIS is a basic molecular biology technique that could be used across several biological disciplines.

Procedure

The MARIS staining and FACS procedure was performed essentially as described in the original publication from the Melton group (Hrvatin et al., 2014) with some modifications (see below):

Human and mouse cortical nephrogenic zone cells were digested from E16.5 embryonic mouse kidneys or 13-18 week fetal human kidneys using 10 mg/ml pancreatin (Sigma, P1625) and 2.5 mg/ml collagenase A (Roche, 11 088 793 001
) enzyme mixture and filtered through 40 µm filter (BD Falcon 352340) as described in Brown et al., 2014.

Cell fixation, washing, permeabilization, and centrifugation were performed as described in Hrvatin et al., 2014 using the following solutions with all subsequent steps performed at 4 C. Fix buffer: 4% PFA (Electron Microscopy Sciences), 0.1% saponin (Sigma-Aldrich 47036) in molecular grade PBS (Ambion) supplemented with 1∶100 RNasin Plus RNase Inhibitor (Promega, N2615) Wash Buffer: PBS containing 0.2% BSA (Gemini Bio-Products), 0.1% saponin (Sigma-Aldrich), 1∶100 RNasin Plus RNase Inihibitor.

SIX2 Primary antibody (Mybiosource, MBS610128) staining of cells was carried out while rocking for overnight at 4 C in staining buffer containing PBS with 1% BSA, 0.1% saponin and 1∶25 RNasin Plus RNase Inhibitor.

Cells were washed and stained with donkey anti-rabbit Alexa 488 (Thermofischer, A-21206) secondary antibody for 45 minutes. Subsequent washing and FACS sorting were performed at a concentration of 5-10 M cells/ml with sort buffer containing PBS, 0.5% BSA, and 1∶25 RNasin Plus RNase Inhibitor.

Cells were sorted on the FACSAria I or II (BD Biosciences) using FACSDiva software. Prior to sorting FACSAria was decontaminated by running RNAse free water through the fluidics for 10 minutes at flow rate 11. Sorting gates were set with reference to negative controls with no primary antibody stain. The sorting efficiency was maintained at above 90%. Cells were collected in tubes that were coated with a small amount of Sort buffer.

RNA isolation

RNA isolation was performed as described in Hrvatin et al., 2014 with the following modifications:

Following FACS, SIX2+ and SIX2- cells were pelleted by centrifugation at 3000 g for 10′ at 4°. Total RNA was isolated using the RecoverAll Total Nucleic Acid Isolation kit (Ambion, AM1975), starting at protease digestion step with protease incubation time of 1 to 3 hours at 50°C and termination at 80 C for precisely 15 minutes. Cell lysates were frozen at −80°C overnight and extracted for RNA according to the manufacturers recommended protocol.

References

Brown, Aaron C., Muthukrishnan, Sree D. & Oxburgh, L. A Synthetic Niche for Nephron Progenitor Cells. Developmental Cell 34, 229-241 doi:10.1016/j.devcel.2015.06.021 (2015).

Hrvatin, S., Deng, F., O’Donnell, C. W., Gifford, D. K. & Melton, D. A. MARIS: method for analyzing RNA following intracellular sorting. PloS one 9, e89459, doi:10.1371/journal.pone.0089459 (2014).

Consortium

(Re)Building a Kidney (RBK) Consortium