Figure 1.
High quality RNA isolation from fixed and stained cells.
(A) Outline of the developed protocol. In vivo or in vitro-derived cells are dispersed, fixed in 4% PFA, permeabilized, stained using standard immunofluorescent antibodies and FACS sorted. Total RNA is isolated using a modified RNA extraction protocol (see methods). (B) RNA was isolated and analyzed from hESC-derived Stage 6 cells before fixation (live) or following fixation, staining and sorting (processed). Simulated electropherogram suggests minimal degradation of total RNA based on the clearly defined 18S and 28S ribosomal RNA bands; RIN value 8.1 for live, 8.0 for processed sample. (C) RNA was isolated from multiple samples across three independent experiments. The average RIN score was 8.3±0.7 (mean±SEM, n = 14) and the average yield 8.35±1.61 pg total RNA per cell (mean±SEM, n = 13).
Figure 2.
Quantitative comparison of live and processed cells.
(A) qRT-PCR on live and processed Stage 6 cells (n = 3) for pancreatic and housekeeping genes. (B) Logarithmic scatter plots of Illumina microarray data between live and processed stage 6 samples show r2 = 0.963±0.005 (mean±SEM, n = 3, r2 determined by Pearson's correlation) correlation for all detected probes (detection p<0.05). Red lines represent 2-fold change. (C) Samples were prepared and paired-end sequenced using TruSeq chemistry on a HiSeq 2000 (Illumina). GENCODE per-gene FPKM values on a logarithmic plot. r2 = 0.97 (Pearson's correlation). Red lines represent 2-fold change. (D) Relative RNA-seq coverage of all annotated transcripts shows 3′ bias in longer length genes. Live and processed RNA-seq read coverage over length-normalized GENCODE transcripts (Live area under the curve 0.133, Processed area under the curve 0.123). Coverage counts were normalized by per-experiment sequencing depth.
Figure 3.
Sorting of insulin-expressing cells from human pluripotent stem cells and adult human islets.
(A) FACS plot of Stage 6 H1-derived cells sorted for insulin and somatostatin. (B) RNA samples were isolated from sort in panel A. RIN scores indicate RNA quality. (C) qRT-PCR of unsorted cells compared to and INS+ SST− cells and INS+ SST+ cells (Paired two-tailed t-test; * p<0.05, **p<0.01). (D) FACS plot of human adult islets sorted for insulin (INS). (B) RNA samples were isolated from sort in panel D. RIN scores indicate RNA quality. (C) qRT-PCR of unsorted islets compared to and INS+ and INS− cells (Paired two-tailed t-test; * p<0.05).