Generation of microRNA Sponge Library
Author: Sebastian Herzog,
date: 04/20/2018,
view: 17,
Q&A: 0
[Abstract] This protocol describes the generation and functional validation of microRNA (miRNA) sponge or decoy constructs. When expressed from a strong promoter, these transcripts can sequester specific miRNA:RISC complexes, thereby resulting in a derepression of endogenous target mRNA. Hence, cells expressing such sponges display a partial or full miRNA ...
Method for CRISPR/Cas9 Mutagenesis in Candida albicans
Authors: Neta Dean and
Henry Ng,
date: 04/20/2018,
view: 18,
Q&A: 0
[Abstract] Candida albicans is the most prevalent and important human fungal pathogen. The advent of CRISPR as a means of gene editing has greatly facilitated genetic analysis in C. albicans. Here, we describe a detailed step-by-step procedure to construct and analyze C. albicans deletion mutants. This protocol uses plasmids that ...
Isolation of Nuclei in Tagged Cell Types (INTACT), RNA Extraction and Ribosomal RNA Degradation to Prepare Material for RNA-Seq
Authors: Mauricio A. Reynoso,
Germain C. Pauluzzi,
Sean Cabanlit,
Joel Velasco,
Jérémie Bazin,
Roger Deal,
Siobhan Brady,
Neelima Sinha,
Julia Bailey-Serres and
Kaisa Kajala,
date: 04/05/2018,
view: 600,
Q&A: 0
[Abstract] Gene expression is dynamically regulated on many levels, including chromatin accessibility and transcription. In order to study these nuclear regulatory events, we describe our method to purify nuclei with Isolation of Nuclei in TAgged Cell Types (INTACT). As nuclear RNA is low in polyadenylated transcripts and conventional pulldown methods would ...
Detection and Analysis of Circular RNAs by RT-PCR
Authors: Amaresh C Panda and
Myriam Gorospe,
date: 03/20/2018,
view: 762,
Q&A: 0
[Abstract] Gene expression in eukaryotic cells is tightly regulated at the transcriptional and posttranscriptional levels. Posttranscriptional processes, including pre-mRNA splicing, mRNA export, mRNA turnover, and mRNA translation, are controlled by RNA-binding proteins (RBPs) and noncoding (nc)RNAs. The vast family of ncRNAs comprises diverse regulatory ...
RNA Cap Methyltransferase Activity Assay
Authors: Jackson B. Trotman and
Daniel R. Schoenberg,
date: 03/20/2018,
view: 318,
Q&A: 0
[Abstract] Methyltransferases that methylate the guanine-N7 position of the mRNA 5’ cap structure are ubiquitous among eukaryotes and commonly encoded by viruses. Here we provide a detailed protocol for the biochemical analysis of RNA cap methyltransferase activity of biological samples. This assay involves incubation of cap-methyltransferase-containing ...
Single-step Precision Genome Editing in Yeast Using CRISPR-Cas9
[Abstract] Genome modification in budding yeast has been extremely successful largely due to its highly efficient homology-directed DNA repair machinery. Several methods for modifying the yeast genome have previously been described, many of them involving at least two-steps: insertion of a selectable marker and substitution of that marker for the intended ...