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Biochemistry

Categories
+ Carbohydrate
+ DNA
+ Lipid
+ Protein
- RNA
miRNA tagging
miRNA targeting
mRNA translation
RNA structure
RNA-protein interaction
- Single-molecule Activity
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Protocols in Past Issues

Preparing and Evaluating the Stability of Therapeutically Relevant Oligonucleotide Duplexes
SI Shreyas G. Iyer
AK Andrea L. Kasinski
Q&A 0 Q&A
4648 Views
Apr 20, 2024

The field of oligonucleotide therapeutics is rapidly advancing, particularly for combating orphan diseases and cancer. However, the intrinsic instability of oligonucleotides, especially RNA, poses a substantial challenge in the face of the harsh conditions encountered intracellularly and in circulation. Therefore, evaluating the stability of oligos in serum is of great significance when developing oligonucleotide therapeutics. This protocol outlines a dependable and reproducible method for preparing oligonucleotide duplexes, coupled with confirmation by gel electrophoresis. Subsequently, the protocol defines a mechanism to assess the stability of the oligo duplexes in serum. This protocol seeks to establish a standardized reference for researchers, enabling them to compare the impact of various modifications on oligo stability and assess the degradation kinetics effectively.

Visualization, Quantification, and Modeling of Endogenous RNA Polymerase II Phosphorylation at a Single-copy Gene in Living Cells
LF Linda S. Forero-Quintero
WR William Raymond
BM Brian Munsky
TS Timothy J. Stasevich
Q&A 0 Q&A
3376 Views
Aug 5, 2022

In eukaryotic cells, RNA Polymerase II (RNAP2) is the enzyme in charge of transcribing mRNA from DNA. RNAP2 possesses an extended carboxy-terminal domain (CTD) that gets dynamically phosphorylated as RNAP2 progresses through the transcription cycle, therefore regulating each step of transcription from recruitment to termination. Although RNAP2 residue-specific phosphorylation has been characterized in fixed cells by immunoprecipitation-based assays, or in live cells by using tandem gene arrays, these assays can mask heterogeneity and limit temporal and spatial resolution. Our protocol employs multi-colored complementary fluorescent antibody-based (Fab) probes to specifically detect the CTD of the RNAP2 (CTD-RNAP2), and its phosphorylated form at the serine 5 residue (Ser5ph-RNAP2) at a single-copy HIV-1 reporter gene. Together with high-resolution fluorescence microscopy, single-molecule tracking analysis, and rigorous computational modeling, our system allows us to visualize, quantify, and predict endogenous RNAP2 phosphorylation dynamics and mRNA synthesis at a single-copy gene, in living cells, and throughout the transcription cycle.


Graphical abstract:



Schematic of the steps for visualizing, quantifying, and predicting RNAP2 phosphorylation at a single-copy gene.


Single-cell qPCR Assay with Massively Parallel Microfluidic System
MP Marta Prieto-Vila
TO Takahiro Ochiya
YY Yusuke Yamamoto
Q&A 0 Q&A
6084 Views
Mar 20, 2020
The single-cell transcriptome is the set of messenger RNA molecules expressed in one cell. It is extremely variable and changes according to external, physical and biochemical conditions. Due to sensitivity shortages, most of genetic studies use bulk samples, providing only the average gene expression. Single-cell technologies have provided a powerful approach to a more detailed understanding of the heterogenic populations and minority cells. However, since it is still a quite novel technique, standardized protocol has to be established. Single-cell qPCR, although partly limited by the number of genes, is relatively simple to analyze. Therefore, its use is accessible without the necessity to recourse to complex bioinformatics analyses. The main steps for single-cell qPCR, as illustrated in this protocol, are composed by single-cell isolation, cell lysate, cDNA reverse-transcription synthesis, amplification for cDNA library generation, and finally, quantitative polymerase chain reaction.

Laser Capture Micro-dissection (LCM) of Neonatal Mouse Forebrain for RNA Isolation
Achira Roy Achira Roy
MD Mei Deng
Kimberly A. Aldinger Kimberly A. Aldinger
Ian A. GLass Ian A. GLass
Kathleen J. Millen Kathleen J. Millen
Q&A 0 Q&A
7801 Views
Jan 5, 2020
Precise and reproducible isolation of desired cell types or layers from heterogeneous tissues is crucial to analyze specific gene profiles and molecular interactions in vivo. Forebrain is the core site of higher functions, like cognition and memory consolidation. It is composed of heterogeneous and distinct cell types, interconnected to form functional neural circuits. Any alteration in the development or function often leads to brain disorders with profound consequences. Thus, precise molecular understanding of forebrain development in normal and diseased scenarios is important. For quantitative studies, most traditional analytical methods require pooling of large cell populations, that results in loss of in vivo tissue integrity and of spatial, molecular and cellular resolution. Laser capture microdissection (LCM) is a fast and extremely precise method of obtaining uncontaminated, homogeneous sets of specific cell types and layers. Our current procedure involves cryo-sectioning and laser microdissection of fresh-frozen mouse forebrains, that are genetically modified and treated with small-molecule therapeutics. Using LCM, specific regions of interest, such as neural layers, cells from adjacent yet distinct subregions within a tissue layer, are obtained under RNase-free conditions. These small cellular cohorts are further used for downstream, high-throughput genomic or transcriptomic assays. Here, we have introduced break-points at multiple stages throughout our protocol. This makes our method simpler and more user-friendly to follow, without compromising on the quality. The current protocol can easily be adapted for different brain regions, as well as for other model organisms/human tissue.

Cloud-point PEG Glass Surfaces for Imaging of Immobilized Single Molecules by Total-internal-reflection Microscopy
ZZ Zhengjian Zhang
SP Sang Ryul Park
AP Alexandros Pertsinidis
AR Andrey Revyakin
Q&A 0 Q&A
13304 Views
Apr 5, 2016
This effective, robust protocol generates glass coverslips coated with biotin-functionalized polyethylene glycol (PEG), making the glass surface resistant to non-specific absorption of biomolecules, and permitting immobilization of biomolecules for subsequent single-molecule tracking of biochemical reactions. The protocol can be completed in one day, and the coverslips can be stored for at least 1 month. We have confirmed that the PEG surfaces prepared according to the protocol are resistant to non-specific adsorption by a wide range of biomolecules (bacterial, mitochondrial, and human transcription factors, DNA, and RNA) and biological buffers.