MARIS: Method for Analyzing RNA following Intracellular Sorting

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.Howard Hughes Medical InstituteHarvard Stem Cell InstituteNational Institutes of Health (U.S.) (grant 2U01DK07247307)National Institutes of Health (U.S.) (grant RL1DK081184)National Institutes of Health (U.S.) (grant 1U01HL10040804

High quality RNA isolation from fixed and stained cells.

<p>(A) Outline of the developed protocol. <i>In vivo</i> or <i>in vitro</i>-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).</p

Quantitative comparison of live and processed cells.

<p>(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.</p

Widespread Occurrence and Genomic Context of Unusually Small Polyketide Synthase Genes in Microbial Consortia Associated with Marine Sponges

Numerous marine sponges harbor enormous amounts of as-yet-uncultivated bacteria in their tissues. There is increasing evidence that these symbionts play an important role in the synthesis of protective metabolites, many of which are of great pharmacological interest. In this study, genes for the biosynthesis of polyketides, one of the most important classes of bioactive natural products, were systematically investigated in 20 demosponge species from different oceans. Unexpectedly, the sponge metagenomes were dominated by a ubiquitously present, evolutionarily distinct, and highly sponge-specific group of polyketide synthases (PKSs). Open reading frames resembling animal fatty acid genes were found on three corresponding DNA regions isolated from the metagenomes of Theonella swinhoei and Aplysina aerophoba. Their architecture suggests that methyl-branched fatty acids are the metabolic product. According to a phylogenetic analysis of housekeeping genes, at least one of the PKSs belongs to a bacterium of the Deinococcus-Thermus phylum. The results provide new insights into the chemistry of sponge symbionts and allow inference of a detailed phylogeny of the diverse functional PKS types present in sponge metagenomes. Based on these qualitative and quantitative data, we propose a significantly simplified strategy for the targeted isolation of biomedically relevant PKS genes from complex sponge-symbiont associations

Sorting of insulin-expressing cells from human pluripotent stem cells and adult human islets.

<p>(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).</p

Genome-Wide High-Throughput Mining of Natural-Product Biosynthetic Gene Clusters by Phage Display

SummaryWe have developed a phage-display method for high-throughput mining of bacterial gene clusters encoding the natural-product biosynthetic enzymes, polyketide synthases (PKSs) and nonribosomal peptide synthetases (NRPSs). This method uses the phosphopantetheinyl transferase activity of Sfp to specifically biotinylate NRPS and PKS carrier-protein domains expressed from a library of random genome fragments fused to a gene encoding a phage coat protein. Subsequently, the biotinylated phages are enriched through selection on streptavidin-coated plates. Using this method, we isolated phage clones from the multiple NRPS and PKS gene clusters encoded in the genomes of Bacillus subtilis and Myxococcus xanthus. Due to the rapid and unambiguous identification of carrier domains, this method will provide an efficient tool for high-throughput cloning of NRPS and PKS gene clusters from many individual bacterial genomes and multigenome environmental DNA