RNA extraction | EPFL Graph Search

RNA extraction is the purification of RNA from biological samples. This procedure is complicated by the ubiquitous presence of ribonuclease enzymes in cells and tissues, which can rapidly degrade RNA. Several methods are used in molecular biology to isolate RNA from samples, the most common of these is guanidinium thiocyanate-phenol-chloroform extraction. The filter paper based lysis and elution method features high throughput capacity. RNA extraction in liquid nitrogen, commonly using a mortar and pestle (or specialized steel devices known as tissue pulverizers) is also useful in preventing ribonuclease activity. RNase contamination The extraction of RNA in molecular biology experiments is greatly complicated by the presence of ubiquitous and hardy RNases that degrade RNA samples. Certain RNases can be extremely hardy and inactivating them is difficult compared to neutralizing DNases. In addition to the cellular RNases that are released there are several RNases that are pre

Live-seq enables temporal transcriptomic recording of single cells

Wanze Chen, Riccardo Dainese, Bart Deplancke, Vincent Roland Julien Gardeux, Pernille Yde Rainer, Wouter Saelens, Magda Zachara

Single-cell transcriptomics (scRNA-seq) has greatly advanced our ability to characterize cellular heterogeneity(1). However, scRNA-seq requires lysing cells, which impedes further molecular or functional analyses on the same cells. Here, we established Live-seq, a single-cell transcriptome profiling approach that preserves cell viability during RNA extraction using fluidic force microscopy(2,3), thus allowing to couple a cell's ground-state transcriptome to its downstream molecular or phenotypic behaviour. To benchmark Live-seq, we used cell growth, functional responses and whole-cell transcriptome read-outs to demonstrate that Live-seq can accurately stratify diverse cell types and states without inducing major cellular perturbations. As a proof of concept, we show that Live-seq can be used to directly map a cell's trajectory by sequentially profiling the transcriptomes of individual macrophages before and after lipopolysaccharide (LPS) stimulation, and of adipose stromal cells pre- and post-differentiation. In addition, we demonstrate that Live-seq can function as a transcriptomic recorder by preregistering the transcriptomes of individual macrophages that were subsequently monitored by time-lapse imaging after LPS exposure. This enabled the unsupervised, genome-wide ranking of genes on the basis of their ability to affect macrophage LPS response heterogeneity, revealing basal Nfkbia expression level and cell cycle state as important phenotypic determinants, which we experimentally validated. Thus, Live-seq can address a broad range of biological questions by transforming scRNA-seq from an end-point to a temporal analysis approach.

NATURE PORTFOLIO2022

Method of preparation of cdna library useful for efficient mrna sequencing and uses thereof

Daniel Alpern, Riccardo Dainese, Mustafa Demir, Bart Deplancke

The invention relates to methods for the preparation of method of preparation of cDNA library based on one or many RNA samples useful for efficient RNA sequencing and uses thereof. The invention further relates to related tools and kits useful in said method.

2022

ORGANOHALIDE RESPIRATION: STUDY OF THE MEMBRANE PROTEIN COMPLEX RESPONSIBLE FOR THE REDUCTION OF HALOGENATED COMPOUNDS

Lorenzo Cimmino, Christof Holliger, Julien Maillard, Adrian Schmid

Organohalide respiration (OHR) is a bacterial anaerobic respiratory process in which halogenated compounds are used as terminal electron acceptors. Desulfitobacterium and Dehalobacter, paradigmatic organohalide-respiring bacteria (OHRB), harbour the pceABCT gene cluster, representing one model system in OHR. While both Dehalobacter restrictus and Desulfitobacterium hafniense strain TCE1 dechlorinate tetrachloroethene (PCE), 3,5-dichlorophenol (DCP) is dechlorinated by D. hafniense strain DCB-2. The relatively high DNA sequence identity (70%) and similar gene composition and regulation between the pceABCT and dcpABCT gene clusters triggered our interest in studying the possible co-transcription of the genes, in establishing stoichiometric relationships between the corresponding proteins, and in trying to decipher the reductive dehalogenase (RDH) protein complex from the cytoplasmic membrane of those bacteria. Gene co-transcription was investigated by a combination of RNA extraction and RT-qPCR, while the stoichiometry of the proteins is analyzed via quantitative proteomics. Clear-Native PAGE technology combined with an in-gel reductive dehalogenase activity assay are established to identify proteins from the RDH complex. The transcriptional analysis conducted on D. hafniense strain DCB-2 showed that dcp gene cluster is actively up-regulated by 3,5-DCP. At protein level, reference peptides were defined for PceA, B, C and T from D. restrictus and D. hafniense strain TCE1 and they will be applied for measuring the relative stoichiometry of the targeted proteins. Furthermore, preliminary results from CN-PAGE analysis suggest that the PCE reductive dehalogenase (PceA) catalytic subunit is part of a ~150 kDa protein complex in D. restrictus. At transcriptional level the results suggest that dcp gene cluster shows an operon structure. However, the possibility of additional promoters regulating the transcription of individual genes within the operon are under investigation. At protein level, the identification of the RDH complex allows us to address the question of the proteins that are associated with PceA.

2019

ORGANOHALIDE RESPIRATION: STUDY OF THE MEMBRANE PROTEIN COMPLEX RESPONSIBLE FOR THE REDUCTION OF HALOGENATED COMPOUNDS

Lorenzo Cimmino, Christof Holliger, Julien Maillard, Adrian Schmid

2019