Molecular Biology

Plant embryos are contained within seeds. Isolating them is crucial when endosperm and seed coat tissues interfere with the study of mutant genetic functions due to differing genotypes between maternal and embryonic tissues. RNA extraction from plant embryonic tissue presents particular challenges due to the high activity of RNases, the composition of the seed, and the risk of RNA degradation. The developmental stage of the embryo is a key aspect of successful isolation and RNA extraction due to the size and amount of tissue. Proper handling during RNA extraction is critical to maintain RNA integrity and prevent degradation. While commercial kits offer various methods for RNA extraction from embryos, homemade protocols provide valuable advantages, including cost-effectiveness and accessibility for labs with limited funding. Here, we present a simple and efficient protocol for extracting RNA from isolated Arabidopsis thaliana embryos at the torpedo/cotyledon stage using a homemade extraction buffer previously reported for styles of Nicotiana alata.

Human astroviruses pose a significant public health threat, especially to children, the elderly, and immunocompromised individuals. Nevertheless, these viruses remain largely understudied, with no approved antivirals or vaccines. This protocol focuses on leveraging reverse genetics (RG) and replicon systems to unravel the biology of MLB genotypes, a key group of neurotropic astroviruses. Using reverse genetics and replicon systems, we identified critical genetic deletions linked to viral attenuation and neurotropism, pushing forward vaccine development. We also uncovered novel replication mechanisms involving ER membrane interactions, opening doors to new antiviral targets. Reverse genetics and replicon systems are essential for advancing our understanding of astrovirus biology, identifying virulence factors, and developing effective treatments and vaccines to combat their growing public health impact.

Transfer RNAs (tRNAs), the essential adapter molecules in protein translation, undergo various post-transcriptional modifications. These modifications play critical roles in regulating tRNA folding, stability, and codon–anticodon interactions, depending on the modified position. Methods for detecting modified nucleosides in tRNAs include isotopic labeling combined with chromatography, antibody-based techniques, mass spectrometry, and high-throughput sequencing. Among these, high-performance liquid chromatography (HPLC) has been a cornerstone technique for analyzing modified nucleosides for decades. In this protocol, we provide a detailed, streamlined approach to purify and digest tRNAs from yeast cells and analyze the resulting nucleosides using HPLC. By assessing UV absorbance spectra and retention times, modified nucleosides can be reliably quantified with high accuracy. This method offers a simple, fast, and accessible alternative for studying tRNA modifications, especially when advanced technologies are unavailable.

Cellular communication relies on the intricate interplay of signaling molecules, which come together to form the cell–cell interaction (CCI) network that orchestrates tissue behavior. Researchers have shown that shallow neural networks can effectively reconstruct the CCI from the abundant molecular data captured in spatial transcriptomics (ST). However, in scenarios characterized by sparse connections and excessive noise within the CCI, shallow networks are often susceptible to inaccuracies, leading to suboptimal reconstruction outcomes. To achieve a more comprehensive and precise CCI reconstruction, we propose a novel method called triple-enhancement-based graph neural network (TENET). The TENET framework has been implemented and evaluated on both real and synthetic ST datasets. This protocol primarily introduces our network architecture and its implementation.

The existence and functional relevance of DNA and RNA G-quadruplexes (G4s) in human cells is now beyond debate, but how did we reach such a level of confidence? Thanks to a panoply of molecular tools and techniques that are now routinely implemented in wet labs. Among them, G4 imaging ranks high because of its reliability and practical convenience, which now makes cellular G4 detection quick and easy; also, because this technique is sensitive and responsive to any G4 modulations in cells, which thus allows gaining precious insights into G4 biology. Herein, we briefly explain what a G4 is and how they can be visualized in human cells; then, we present the strategy we have been developing for several years now for in situ click G4 imaging, which relies on the use of biomimetic G4 ligands referred to as TASQs (for template-assembled synthetic G-quartets) and is far more straightforward and modular than classically used immunodetection methods. We thus show why and how to illuminate G4s with TASQs and provide a detailed, step-by-step methodology (including the preparation of the materials, the methodology per se, and a series of notes to address any possible pitfalls that may arise during the experiments) to make G4 imaging ever easier to operate.

Cucumber (Cucumis sativus) trichomes play a critical role in resisting external biological and abiotic stresses. Glandular trichomes are particularly significant as they serve as sites for the synthesis and secretion of secondary metabolites, while non-glandular trichomes are pivotal for determining the appearance quality of cucumbers. However, current methods for separating trichomes encounter challenges such as low efficiency and insufficient accuracy, limiting their applicability in multi-omics sequencing studies. This protocol introduces an efficient system designed for the precise separation of glandular and non-glandular trichomes from cucumber fruit. The process begins with the pre-cooling of sorbitol buffer or ethanol solution and the RNA-free treatment of laboratory supplies, followed by sterilization and pre-cooling. After filling glass bottles with pre-cooling buffer and glass beads, cucumber ovaries are then placed in the glass bottles and the trichome is harvested by bead-beating method. The separation process involves sequential filtration through various steel sieves and centrifugation to separate trichomes. The separated trichomes obtained from this method are well-suited for subsequent multi-omics sequencing analyses. This protocol achieved high precision in separating glandular and non-glandular trichomes, significantly enhancing the efficiency of separation and sample collection processes. This advancement not only addresses existing limitations but also facilitates comprehensive studies aimed at exploring the genetic and biochemical diversity present within cucumber trichomes, thereby opening avenues for broader agricultural and biological research applications.

MicroRNAs (miRNAs) are small, non-coding RNAs that play pivotal roles in gene regulation; they are increasingly recognized as vital biomarkers for various diseases, notably cancer. Conventional methods for miRNA detection, such as quantitative PCR and microarray analysis, often entail intricate sample preparation and lack the requisite sensitivity to detect low-abundance miRNAs like miRNA-21. This protocol presents an innovative approach that combines branched hybridization chain reaction (bHCR) with DNAzyme technology for the precise detection of miRNA-21. The bHCR amplifies the target signal through a branched structure, while the DNAzyme boosts detection sensitivity through catalytic cleavage, enabling swift and specific identification of miRNA-21. This dual amplification strategy offers a highly sensitive, specific, and rapid alternative to traditional techniques, making it particularly well-suited for early-stage disease diagnosis.

Genome-wide gene expression analysis is a commonly used method to quantitatively examine the transcriptional signature of any tissue or cell state. Standard bulk cell RNA sequencing (RNA-seq) quantifies RNAs in the cells of the tissue type of interest through massive parallel sequencing of cDNA synthesized from the cellular RNA. The subsequent analysis of global RNA expression and normalization of RNA expression levels between two or more samples generally assumes that cells from all samples produce equivalent amounts of RNA per cell. This assumption may be invalid in cells where MYC or MYCN expression levels are markedly different and thus, overall mRNA expression per cell is altered. Here, we describe an approach for RNA-seq analysis of MYCN-amplified neuroblastoma cells during treatment with retinoic acid, which causes dramatic downregulation of MYCN expression and induces growth arrest and differentiation of the cells. Our procedure employs spiked-in RNA standards added in ratio to the number of cells in each sample prior to RNA extraction. In the analysis of differential gene expression, the expression level of each gene is standardized to the spiked-in RNA standard to accurately assess gene expression levels per cell in conditions of high and low MYCN expression. Our protocol thus provides a step-by-step experimental approach for normalizing RNA-seq expression data on a per-cell-number basis, allowing accurate assessment of differential gene expression in cells expressing markedly different levels of MYC or MYCN.

Regulation of gene expression in response to various biological processes, including extracellular stimulation and environmental adaptation, requires nascent mRNA synthesis and translation. Simultaneous analysis of the coordinated regulation of dynamic mRNA synthesis and translation using the same experiment remains a major challenge in the field. Here, we describe a step-by-step protocol for the simultaneous measurement of transcription of nascent mRNA and its translation at the gene level during the acute unfolded protein response (UPR) in HEK293 cells by combining 4-thiouridine metabolic mRNA labeling with translational ribosome affinity purification (TRAP) using a monoclonal antibody against evolutionarily conserved ribosomal P-stalk proteins (P-TRAP). Since P-TRAP captures full-length RNAs bound to ribosomes, it is compatible with 3' mRNA-seq, which analyzes the uridine-rich 3' UTRs of polyadenylated RNAs, allowing robust quantification of T>C conversions. Our nascent P-TRAP (nP-TRAP) method, in which P-TRAP is combined with metabolic mRNA labeling, can serve as a simple and powerful tool to analyze the coordinated regulation of transcription and translation of individual genes in cultured cells.

Single-cell transcriptomic analyses have emerged as very powerful tools to query the gene expression changes at the single-cell level in physiological and pathological conditions. The quality of the analysis is heavily dependent on tissue digestion protocols, with the goal of preserving thousands of single live cells to submit to the subsequent processing steps and analysis. Multiple digestion protocols that use different enzymes to digest the tissues have been described. Harsh digestion can damage certain cell types, but this might be required to digest especially fibrotic tissue as in our experimental condition. In this paper, we summarize a collagenase type I digestion protocol for preparing the single-cell suspension from fibrovascular tissues surgically removed from patients with proliferative diabetic retinopathy (PDR) for single-cell RNA sequencing (scRNA-Seq) analyses. We also provide a detailed description of the data analysis that we implemented in a previously published study.