Stougaardfisher3948

In order to validate fungal miRNAs that were imported into the host cell, we developed a straightforward method to isolate protoplasts from tomato roots infected by Fusarium oxysporum f.sp. lycopersici using enzymatic digestion.Over the past few decades, various techniques have been developed and optimized for the accurate measurement of RNA abundance in cells or tissues. These methods have been instrumental in gaining insight in complex systems such as host-symbiont associations. The pea aphid model has recently emerged as a powerful and experimentally tractable system for studying symbiotic relationships and it is the subject of a growing number of molecular studies. Nevertheless, the lack of standardized protocols for the collection of bacteriocytes, the specialized host cells harboring the symbionts, has limited its use. This chapter provides a simple, step-by-step dissection protocol for the rapid isolation of aphid bacteriocytes. We then describe an adapted protocol for efficient extraction and purification of bacteriocyte RNA that can be used for most downstream transcriptomic analyses.Northern analysis is a conventional but gold standard method for detection and quantification of gene expression changes. It not only detects the presence of a transcript but also indicates size and relative comparison of transcript abundance on a single membrane. In recent years it has been aptly adapted to validate and study the size and expression of small noncoding RNAs. Here, we describe protocols employed in our laboratory for conventional northern analysis with total RNA/mRNA to study gene expression and validation of small noncoding RNAs using low molecular weight fraction of RNAs. A brief account on the recent advancements for improving the sensitivity and efficiency of northern blot detection is also included in this chapter.MicroRNAs (miRNAs) play important roles in development in plants, and some miRNAs show developmentally regulated organ- and tissue-specific expression patterns. learn more Therefore, in situ detection of mature miRNAs is important for understanding the functions for both miRNAs and their targets. The construction of promoter-reporter fusions and examination of their in planta expression has been widely used and the results obtained thus far are rather informative; however, in some cases, the length of promoter that contains entire regulatory elements is difficult to determine. In addition, traditional in situ hybridization with the antisense RNA fragment as the probe usually fails to detect miRNAs, because the mature miRNAs are too short (~21-nucleotides) to exhibit stable hybridization signals. In recent years, the Locked nucleic acid (LNA) modified DNA probe has been successfully used in animals and plants to detect small RNAs. Here, we describe a modified protocol using LNA-modified DNA probes to detect mature miRNAs in plant tissues, including the design of LNA probes and detailed steps for the in situ hybridization experiment, using Arabidopsis miR165 as an example.Chloroplasts are essential semiautonomous plant organelles responsible for photosynthesis, which generates sugars and oxygen vital for the entire biosphere. Additionally, chloroplasts regulate energy production, metabolite synthesis, and stress responses in plants and algae. Chloroplasts possess a notably complex RNA metabolism that includes RNA processing, editing, splicing, and regulation by various RNA-binding proteins. Highly purified chloroplasts, free of nuclear/cytoplasmic contaminants are desirable when studying chloroplast RNA metabolism. Here, we describe an efficient protocol to obtain highly purified chloroplasts for RNA analysis.Next Generation Sequencing (NGS) is becoming a routine experimental technology. It has been a great success in recent years to profile small-RNA species using NGS. Indeed, a large quantity of small-RNA profiling data has been generated from NGS, and computational methods have been developed to process and analyze NGS data for the purpose of identification of novel and expressed small noncoding RNAs and analysis of their roles in nearly all biological processes and pathways in eukaryotes. We discuss here the computational procedures and major steps for identification of microRNAs and natural antisense transcript-originated small interfering RNAs (nat-siRNAs) from NGS small-RNA profiling data.MicroRNAs (miRNAs) are small RNAs, that bind to mRNA targets and regulate their translation. Functional study of miRNAs and exploration of their utility as disease markers require miRNA extraction from biological samples, which contain large amounts of interfering compounds for downstream RNA identification and quantification. The most common extraction methods employ either silica columns or TRIzol reagent, but these approaches afford low recovery for small RNAs, possibly due to their short strand lengths. Here, we describe the fabrication of titanium dioxide nanofibers and the optimal extraction conditions to improve miRNA recovery from biological buffers, cell lysate, and serum.The luciferase reporter assay is a widely used tool to study the cis and trans factors controlling regulation of gene expression. In this assay, regulatory elements can be fused to the luciferase gene, and as a result effect protein output by changing rates of transcription, rates of translation, or mRNA stability. This protocol focuses on probing the function of RNA-binding proteins (RBPs) through their interactions with the 3' untranslated region (UTR), thus examining gene expression regulation on the mRNA level. Assessment of 3' UTR sequence requirements, as well as single and co-regulatory roles of RBPs in regulation of mRNAs will be discussed.The advancement of transcriptomic studies in plant parasitic nematodes will greatly benefit from the development of single-nematode RNA-seq methods. Since many plant parasitic nematodes are obligate parasites, it is often difficult to efficiently obtain sufficient amounts of nematodes for transcriptomic studies. Here we have adapted SMART-Seq2 for single-nematode RNA-seq requiring only an individual nematode for a sample replicate. This protocol provides a detailed step-by-step procedure of the RNA-seq workflow starting from lysis of the nematode to quantification of transcripts using a user-friendly online platform.