Molecular Biology


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Protocols in Current Issue
0 Q&A 436 Views Mar 20, 2023

E-cigarette (E-cig) inhalation affects health status by modulating inflammation profiles in several organs, including the brain, lung, heart, and colon. The effect of flavored fourth-generation pod-based E-cigs (JUUL) on murine gut inflammation is modulated by both flavor and exposure period. Exposure of mice to JUUL mango and JUUL mint for one month upregulated inflammatory cytokines, particularly TNF-α, IL-6, and Cxcl-1 (IL-8). JUUL Mango effects were more prominent than those incurred by JUUL Mint after one month of exposure. However, JUUL Mango reduced the expression of colonic inflammatory cytokines after three months of exposure. In this protocol, we detail the process of RNA isolation from the mouse colon and the use of extracted RNA in profiling the inflammatory milieu. Efficient RNA extraction from the murine colon is the most important step in the evaluation of inflammatory transcripts in the colon.

0 Q&A 246 Views Mar 20, 2023

Polysome profiling by sucrose density gradient centrifugation is commonly used to study the overall degree of translation (messenger RNA to protein synthesis). Traditionally, the method begins with synthesis of a 5–10 mL sucrose gradient onto which 0.5–1 mL of cell extract is layered and centrifuged at high speed for 3–4 h in a floor-model ultracentrifuge. After centrifugation, the gradient solution is passed through an absorbance recorder to generate a polysome profile. Ten to twelve fractions (0.8–1 mL each) are collected for isolating different RNA and protein populations. The overall method is tedious and lengthy (6–9 h), requires access to a suitable ultracentrifuge rotor and centrifuge, and requires a substantial amount of tissue material, which can be a limiting factor. Moreover, there is often a dilemma over the quality of RNA and protein populations in the individual fractions due to the extended experiment times. To overcome these challenges, here we describe a miniature sucrose gradient for polysome profiling using Arabidopsis thaliana seedlings that takes ~1 h centrifugation time in a tabletop ultracentrifuge, reduced gradient synthesis time, and also less tissue material. The protocol described here can be easily adapted to a wide variety of organisms and polysome profiling of organelles, such as chloroplasts and mitochondria.


Key Features

• Mini sucrose gradient for polysome profiling that requires less than half the processing time vs. traditional methods.

• Reduced starting tissue material and sample volume for sucrose gradients.

• Feasibility of RNA and protein isolation from polysome fractions.

• Protocol can be easily modified to a wide variety of organisms (and even polysome profiling of organelles, such as chloroplast and mitochondria).


Graphical Overview



Figure 1. Graphical overview of polysome profiling using mini sucrose gradient. A. One milliliter each of 15% (w/v) and 50% (w/v) sucrose gradient solution is added to the individual chambers of the gradient maker. While mixing with a small magnetic stirrer in the 50% solution chamber, base station knob is turned to open position, allowing sucrose gradient solution to slowly flow through the outlet into a 2.2 mL gradient tube. After centrifugation at 50,000 rpm (213,626.2 × g) in a swinging bucket rotor for 70 min at 4 °C, the gradient tube is stored at 4 °C for the next steps. B. Cell extract from 12-day-old vertically grown Arabidopsis thaliana seedlings is centrifuged twice and 100 µL of supernatant is gently layered on the pre-made sucrose gradient from step A. After centrifugation as described in step A, polysome profile is obtained by feeding the gradient solution through an absorbance recorder (A254 nm). Eight (200 µL) fractions are collected for RNA and protein isolation.
Protocols in Past Issues
0 Q&A 118 Views Mar 5, 2023

Telomeres are structures that cap the ends of linear chromosomes and play critical roles in maintaining genome integrity and establishing the replicative lifespan of cells. In stem and cancer cells, telomeres are actively elongated by either telomerase or the alternative lengthening of telomeres (ALT) pathway. This pathway is characterized by several hallmark features, including extrachromosomal C-rich circular DNAs that can be probed to assess ALT activity. These so-called C-circles are the product of ALT-associated DNA damage repair processes and simultaneously serve as potential templates for iterative telomere extension. This bifunctional nature makes C-circles highly sensitive and specific markers of ALT. Here, we describe a C-circle assay, adapted from previous reports, that enables the quantitation of C-circle abundance in mammalian cells subjected to a wide range of experimental perturbations. This protocol combines the Quick C-circle Preparation (QCP) method for DNA isolation with fluorometry-based DNA quantification, rolling circle amplification (RCA), and detection of C-circles using quantitative PCR. Moreover, the inclusion of internal standards with well-characterized telomere maintenance mechanisms (TMMs) allows for the reliable benchmarking of cells with unknown TMM status. Overall, our work builds upon existing protocols to create a generalizable workflow for in vitro C-circle quantitation and ascertainment of TMM identity.

0 Q&A 149 Views Mar 5, 2023

Malaria molecular surveillance has great potential to support national malaria control programs (NMCPs), informing policy for its control and elimination. Here, we present a new three-day workflow for targeted resequencing of markers in 13 resistance-associated genes, histidine rich protein 2 and 3 (hrp2&3), a country (Peru)-specific 28 SNP-barcode for population genetic analysis, and apical membrane antigen 1 (ama1), using Illumina short-read sequencing technology. The assay applies a multiplex PCR approach to amplify all genomic regions of interest in a rapid and easily standardizable procedure and allows simultaneous amplification of a high number of targets at once, therefore having great potential for implementation into routine surveillance practice by NMCPs. The assay can be performed on routinely collected filter paper blood spots and can be easily adapted to different regions to investigate either regional trends or in-country epidemiological changes.

0 Q&A 551 Views Feb 20, 2023

Interactions between RNA-binding proteins and RNA molecules are at the center of multiple biological processes. Therefore, accurate characterization of the composition of ribonucleoprotein complexes (RNPs) is crucial. Ribonuclease (RNase) for mitochondrial RNA processing (MRP) and RNase P are highly similar RNPs that play distinct roles at the cellular level; as a consequence, the specific isolation of either of these complexes is essential to study their biochemical function. Since their protein components are nearly identical, purification of these endoribonucleases using protein-centric methods is not feasible. Here, we describe a procedure employing an optimized high-affinity streptavidin-binding RNA aptamer, termed S1m, to purify RNase MRP free of RNase P. This report details all steps from the RNA tagging to the characterization of the purified material. We show that using the S1m tag allows efficient isolation of active RNase MRP.

0 Q&A 292 Views Feb 5, 2023

Nucleotide excision repair (NER) removes a wide variety of structurally unrelated lesions from the genome, including UV-induced photolesions such as 6–4 pyrimidine–pyrimidone photoproducts (6-4PPs) and cyclobutane pyrimidine dimers (CPDs). NER removes lesions by excising a short stretch of single-stranded DNA containing the damaged DNA, leaving a single-stranded gap that is resynthesized in a process called unscheduled DNA synthesis (UDS). Measuring UDS after UV irradiation in non-dividing cells provides a measure of the overall NER activity, of which approximately 90% is carried out by the global genome repair (GGR) sub pathway. Here, we present a protocol for the microscopy-based analysis and quantification of UDS as a measurement for GGR activity. Following local UV-C irradiation, serum-starved human cells are supplemented with the thymidine analogue 5-ethynyl-2'-deoxyuridine (EdU), which is incorporated into repair patches following NER-dependent dual incision. The incorporated nucleotide analogue is coupled to a fluorophore using Click-iT chemistry, followed by immunodetection of CPD photolesions to simultaneously visualize both signals by fluorescence microscopy. Accompanying this protocol is a custom-built ImageJ plug-in to analyze and quantify UDS signals at sites of CPD-marked local damage. The local UDS assay enables an effective and sensitive fluorescence-based quantification of GGR activity in single cells with application in basic research to better understand the regulatory mechanism in NER, as well as in diagnostics to characterize fibroblasts from individuals with NER-deficiency disorder.


Graphical abstract


0 Q&A 168 Views Feb 5, 2023

Chemical modifications on RNA play important roles in regulating its fate and various biological activities. However, the impact of RNA modifications varies depending on their locations on different transcripts and cells/tissues contexts; available tools to dissect context-specific RNA modifications are still limited. Herein, we report the detailed protocol for using a chemically inducible and reversible platform to achieve site-specific editing of the chosen RNA modification in a temporally controlled manner by integrating the clustered regularly interspaced short palindromic repeats (CRISPR) technology and the abscisic acid (ABA)-based chemically induced proximity (CIP) system. The procedures were demonstrated using the example of inducible and reversible N6-methyladenosine (m6A) editing and the evaluation of its impact on RNA properties with ABA addition and reversal with the control of ABA or light.

0 Q&A 454 Views Feb 5, 2023

Single-nucleus RNA sequencing (snRNA-seq) provides a powerful tool for studying cell type composition in heterogenous tissues. The liver is a vital organ composed of a diverse set of cell types; thus, single-cell technologies could greatly facilitate the deconvolution of liver tissue composition and various downstream omics analyses at the cell-type level. Applying single-cell technologies to fresh liver biopsies can, however, be very challenging, and snRNA-seq of snap-frozen liver biopsies requires some optimization given the high nucleic acid content of the solid liver tissue. Therefore, an optimized protocol for snRNA-seq specifically targeted for the use of frozen liver samples is needed to improve our understanding of human liver gene expression at the cell-type resolution. We present a protocol for performing nuclei isolation from snap-frozen liver tissues, as well as guidance on the application of snRNA-seq. We also provide guidance on optimizing the protocol to different tissue and sample types.

0 Q&A 219 Views Feb 5, 2023

In addition to cytosolic protein synthesis, mitochondria also utilize another translation system that is tailored for mRNAs encoded in the mitochondrial genome. The importance of mitochondrial protein synthesis has been exemplified by the diverse diseases associated with in organello translation deficiencies. Various methods have been developed to monitor mitochondrial translation, such as the classic method of labeling newly synthesized proteins with radioisotopes and the more recent ribosome profiling. However, since these methods always assess the average cell population, measuring the mitochondrial translation capacity in individual cells has been challenging. To overcome this issue, we recently developed mito-fluorescent noncanonical amino acid tagging (FUNCAT) fluorescence-activated cell sorting (FACS), which labels nascent peptides generated by mitochondrial ribosomes with a methionine analog, L-homopropargylglycine (HPG), conjugates the peptides with fluorophores by an in situ click reaction, and detects the signal in individual cells by FACS equipment. With this methodology, the hidden heterogeneity of mitochondrial translation in cell populations can be addressed.

0 Q&A 271 Views Jan 20, 2023

In this study, we introduce a detailed protocol for the preparation of DNA-assembled GRS-DNA-copper sulfide (CuS) nanodandelion, a multifunctional theranostics nanoparticle. Using transmission electron microscope (TEM) and dynamic light scattering techniques, we characterize the physicochemical property of DNA-assembled GRS-DNA-CuS nanodandelions and their dissociation property after the first near-infrared (NIR) light irradiation. In addition, we systematically monitor the processes of tumor accumulation and uniform intratumoral distribution (UITD) of ultrasmall CuS photothermal agents (PAs), which are dissociated from GRS-DNA-CuS nanodandelions, by Raman imaging and photoacoustic imaging, respectively. The UITD of the dissociated ultrasmall CuS PAs can enhance the therapeutic efficiency of photothermal treatment under the second NIR light irradiation. Overall, this protocol provides a powerful tool to achieve UITD of PAs by explosively breaking the hydrogen bonds of DNA in GRS-DNA-CuS nanodandelions under NIR light irradiation. We expect DNA-assembled nanotheranostics to serve as a robust platform for a variety of biomedical applications related to photothermal therapy in the oncology field. This protocol can increase experimental reproducibility and contribute to efficient theranostics nanomedicine.

0 Q&A 383 Views Jan 5, 2023

Utilizingresources available from the mother's body to guarantee healthy offspring growth is the fundamental reproductive strategy. Recently, we showed that a class of the largest extracellular vesicles known as exophers, which are responsible for the removal of neurotoxic components from neurons (Melentijevic et al., 2017) and damaged mitochondria from cardiomyocytes (Nicolás-Ávila et al., 2020), are released by the Caenorhabditis elegans hermaphrodite body wall muscles (BWM), to support embryonic growth (Turek et al., 2021). Employing worms expressing fluorescent reporters in BWM cells, we found that exopher formation (exophergenesis) is sex-specific and fertility-dependent. Moreover, exophergenesis is regulated by the developing embryo in utero, and exophers serve as transporters for muscle-generated yolk proteins, which can be used to nourish the next generation. Given the specific regulation of muscular exophergenesis, and the fact that muscle-generated exophers are much larger than neuronal ones and have different targeting, their identification and quantification required a modified approach from that designed for neuronal-derived exophers (Arnold et al., 2020). Here, we present a methodology for assessing and quantifying muscle-derived exophers that can be easily extended to determine their function and regulation in various biological contexts.


Graphical abstract





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