Molecular Biology


    Protocols in Current Issue
    Adapting the Smart-seq2 Protocol for Robust Single Worm RNA-seq
    [Abstract] Most nematodes are small worms that lack enough RNA for regular RNA-seq protocols without pooling hundred to thousand of individuals. We have adapted the Smart-seq2 protocol in order to sequence the transcriptome of an individual worm. While developed for individual Steinernema carpocapsae and Caenorhabditis elegans larvae as ...
    RNA Capping by Transcription Initiation with Non-canonical Initiating Nucleotides (NCINs): Determination of Relative Efficiencies of Transcription Initiation with NCINs and NTPs
    Authors:  Jeremy G. Bird, Bryce E. Nickels and Richard H. Ebright, date: 06/20/2017, view: 1970, Q&A: 0
    [Abstract] It recently has been established that adenine-containing cofactors, including nicotinamide adenine dinucleotide (NAD+), reduced nicotinamide adenine dinucleotide (NADH), and 3’-desphospho-coenzyme A (dpCoA), can serve as ‘non-canonical initiating nucleotides’ (NCINs) for transcription initiation by bacterial and eukaryotic cellular RNA ...
    Single-molecule RNA Fluorescence in situ Hybridization (smFISH) in Caenorhabditis elegans
    [Abstract] Single-molecule RNA fluorescence in situ hybridization (smFISH) is a technique to visualize individual RNA molecules using multiple fluorescently-labeled oligonucleotide probes specific to the target RNA (Raj et al., 2008; Lee et al., 2016a). We adapted this technique to visualize RNAs in the C. elegans whole ...
    Single-cell Visualization of Chromosome Transcriptional Territories by RNA-paint
    Authors:  Céline Vallot and Claire Rougeulle, date: 09/05/2016, view: 2925, Q&A: 0
    [Abstract] We developed a FISH-based method to directly assess chromosome-wide transcriptional activity, thereby enabling the visualization of the actively transcribed fraction of a chromosome at the single-cell level. We applied this method to probe the activity of X-chromosomes and its instability in the context of human embryonic stem cells and cancer ...
    In vitro Transcription (IVT) and tRNA Binding Assay
    Author:  Sonja MK Schoenfelder, date: 09/20/2014, view: 5574, Q&A: 0
    [Abstract] This protocol describes the coupling of (i) “live” in vitro RNA transcription with (ii) binding by a radiolabeled, pre-formed tRNA followed by native gel electrophoresis and phosphorimager scan to visualize the complex. The necessity arose from the stable structure that one RNA forms in the absence of its interaction partner. The T-box ...
    Single-cell Gene Expression Profiling of Mouse Stem Cells With Fluidigm BiomarkTM Dynamic Array
    Author:  Ana Sevilla, date: 05/05/2013, view: 9654, Q&A: 3
    [Abstract] This protocol describes how to use Fluidigm BiomarkTM 96.96 dynamic arrays for high-throughput expression profiling from single mouse stem cells, assaying up to 96 independent samples with up to 96 quantitative PCR (qPCR) probes (equivalent to 9,216 reactions) in a single experiment. This Dynamic Array contains a network of microfluidic ...
    Isolation of Epithelial Cells from Mouse Gastrointestinal Tract for Western Blot or RNA Analysis
    Authors:  Maged Zeineldin and Kristi Neufeld, date: 11/20/2012, view: 11377, Q&A: 0
    [Abstract] The gastrointestinal (GI) tract is lined by a single layer of epithelial cells which function in secretion, absorption, and digestion. In addition, most GI tract tumors develop from epithelial cells (carcinomas). This protocol describes isolation of the surface epithelium from the underlying stroma, muscular layer and submucosa in the GI tract. In ...
    Polyadenylated RNA Sampling
    Author:  Eliane Hajnsdorf, date: 09/05/2012, view: 5029, Q&A: 0
    [Abstract] Polyadenylation is a post-transcriptional modification of RNA occurring in prokaryotes, eukaryotes and organelles. However, the function and extent of bacterial polyadenylation are in marked contrast to those of eukaryotic poly(A) tails. In fact, the long poly(A) tails of eukaryotic mRNAs play an important role in their exportation to the ...
    Synthesis of 5’ end-labeled RNA
    Author:  Harald Putzer, date: 07/20/2012, view: 7112, Q&A: 2
    [Abstract] 5’ end-labeled RNA molecules are useful substrates to analyse the endo- and exonucleolytic activities of various ribonucleases. Here two protocols are given to synthesize P32 labeled RNAs with a 5’ PPP or 5’ P moiety. 5’ exoribonucleases generally do not work on 5’ PPP RNA and require a 5’ P substrate. The activity of certain ...
    Primer Extension Analysis Using AMV Reverse Transcriptase
    Author:  Harald Putzer, date: 07/20/2012, view: 5577, Q&A: 0
    [Abstract] Primer extension analysis is a useful method to determine the transcription start point or a processing site on an RNA molecule. It can also allow a quantitative measurement of an RNA species.