Northern blot

Small RNAs play important roles in regulation of plant development and response to various stresses. Northern blot is an important technique in small RNA research. Isotope- and biotin- (or digoxigenin) labeled probes are frequently used in small RNA northern blot. However, isotope-based probe is limited by strict environmental regulation and availability in many places in the world while biotin-based probe is usually suffered from low sensitivity. In this study, we developed a T4 DNA polymerase-based method for incorporation of a cluster of 33 biotin-labeled C in small RNA probe (T4BC33 probe)...

Polymerases encoded by segmented negative-strand RNA viruses cleave 5'-m7G-capped host transcripts to prime viral mRNA synthesis ("cap-snatching") to generate chimeric RNA, and trans-splicing occurs between viral and cellular transcripts. Bombyx mori cytoplasmic polyhedrosis virus (BmCPV), an RNA virus belonging to Reoviridae, is a major pathogen of silkworm (B. mori). The genome of BmCPV consists of 10 segmented double-stranded RNAs (S1-S10) from which viral RNAs encoding a protein are transcribed. In this study, chimeric silkworm-BmCPV RNAs, in which the sequence derived from the silkworm transcript could fuse with both the 5' end and the 3' end of viral RNA, were identified in the midgut of BmCPV-infected silkworms by RNA_seq and further confirmed by RT-PCR and Sanger sequencing...

Lyme borreliosis (LB) is caused by the transmission of Borrelia burgdorferi s.l. from ticks to humans. Climate affects tick abundance, and climate change is projected to promote shifts in abundance in Europe, potentially increasing human exposure. We analyzed serum samples collected between the years 2014-2019 from German National Cohort (NAKO) participants at four study sites (Augsburg, Berlin, Hanover, Münster) for immunoglobulin G (IgG) and immunoglobulin M (IgM) antibodies using an enzyme-linked immunosorbent assay (ELISA) and line blot immunoassay as confirmatory test for positive and equivocal ELISA samples...

Here, we employ coelution experiments and far-western blotting to identify stable interactions between the main components of the B. subtilis degradosome and the small proteins SR1P and SR7P. Our data indicate that B. subtilis has a degradosome comprising at least RNases Y and PnpA, enolase, phosphofructokinase, glycerol-3-phosphate dehydrogenase GapA, and helicase CshA that can be co-purified without cross-linking. All interactions were corroborated by far-western blotting with proteins purified from E. coli...

Mycobacterium avium subsp. paratuberculosis (MAP) causes bovine paratuberculosis (PTB). PTB is responsible for significant economic losses in dairy herds around the word. PTB control programs that rely on testing and culling of test-positive cows have been developed. Current diagnostics, such as ELISA for detecting MAP antibodies in serum samples and PCR detecting MAP DNA in feces, have inadequate sensitivity for detecting subclinical animals. Innovative "omics" technologies such as next-generation sequencing (NGS) technology-based RNA-sequencing (RNA-Seq), proteomics and metabolomics can be used to find host biomarkers...

MicroRNAs (miRNAs) are 21-25 nucleotide long non-coding ribonucleic acids that modulate gene expression by degrading transcripts or inhibiting translation. The miRNA miR-128, originally thought to be brain-specific, was later also found in immune cells. To identify a valuable immune cell model system to modulate endogenous miR-128 amounts and to validate predicted miR-128 target mRNAs in B cells, we first investigated miR-128 expression using Northern blot analysis in several cell lines representing different stages of B cell development...

miRNAs play critical roles in the regulation of plant growth and development by cleaving mRNA or repressing transcription. In our previous study, miR5504 with unknown functions was captured by small RNA sequencing. Here, the function and characters of miR5504 were extensively analyzed using CRISPR/Cas9, overexpression strategy, Northern blot, cytological analysis, and transcriptomics analysis. We found that the dwarf phenotype of mir5504 mutants (mir5504-1 and mir5504-2) appeared on 35-day seedlings and became more apparent at the mature stage...

The extraction of high-quality RNA from kenaf is essential for genetic and molecular biology research. However, the presence of high levels of polysaccharide and polyphenol compounds in kenaf poses challenges for RNA isolation. We proposed a simplified, time-saving and cost-effective method for isolating high quantities of RNA from various kenaf tissues. This method exhibited superior efficiency in RNA isolation compared with the conventional cetyltrimethylammonium bromide method and demonstrated greater adaptability to different samples than commercial kits...

This report describes a chemiluminescence-based detection method for RNAs on northern blots, designated Chemi-Northern. This approach builds on the simplicity and versatility of northern blotting, while dispensing of the need for expensive and cumbersome radioactivity. RNAs are first separated on denaturing gel electrophoresis, transferred to a nylon membrane, and then hybridized to a biotinylated RNA or DNA antisense probe. Streptavidin conjugated with horseradish peroxidase and enhanced chemiluminescence substrate are then used to detect the probe bound to the target RNA...

Autophagy is a lysosomal degradation process known as autophagic flux, involving the engulfment of damaged proteins and organelles by double-membrane autophagosomes. It comprises microautophagy, chaperone-mediated autophagy (CMA), and macroautophagy. Macroautophagy consists of three stages: induction, autophagosome formation, and autolysosome formation. Atg8-family proteins are valuable for tracking autophagic structures and have been widely utilized for monitoring autophagy. The conversion of LC3 to its lipidated form, LC3-II, served as an indicator of autophagy...

BACKGROUND: In addition to the well-known coronavirus genomes and subgenomic mRNAs, the existence of other coronavirus RNA species, which are collectively referred to as noncanonical transcripts, has been suggested; however, their biological characteristics have not yet been experimentally validated in vitro and in vivo. METHODS: To comprehensively determine the amounts, species and structures of noncanonical transcripts for bovine coronavirus in HRT-18 cells and mouse hepatitis virus A59, a mouse coronavirus, in mouse L cells and mice, nanopore direct RNA sequencing was employed...

The poly-adenosine, or poly(A) tail, plays key roles in controlling the stability and translation of messenger RNAs in all eukaryotes, and, as such, facile assays that can measure poly(A) length are needed. This chapter describes an approach that couples RNase H-mediated cleavage of an RNA of interest with high-resolution denaturing gel electrophoresis and northern blot-based detection. Major advantages of this method include the ability to directly measure the abundance of any RNA and the length of its poly(A) tail without amplification steps...

The A1762T/G1764A mutations, one of the most common mutations in the hepatitis B virus basal core promoter, are associated with the progression of chronic HBV infection. However, effects of these mutations on HBV replication remains controversial. This study aimed to systematically investigate the effect of the mutations on HBV replication and its underlying mechanisms. Using the prcccDNA/pCMV-Cre recombinant plasmid system, a prcccDNA-A1762T/G1764A mutant plasmid was constructed. Compared with wild-type HBV, A1762T/G1764A mutant HBV showed enhanced replication ability with higher secreted HBV DNA and RNA levels, while Southern and Northern blot indicated higher intracellular levels of relaxed circular DNA, single-stranded DNA, and 3...

Two endornaviruses, Phytophthora endornavirus 2 (PEV2) and Phytophthora endornavirus 3 (PEV3), have been discovered in pathogens targeting asparagus. In this study, we analyzed the nick structure in the RNA genomes of PEV2 and PEV3 in the host oomycetes. Northern blot hybridization using positive and negative strand-specific RNA probes targeting the 5' and 3' regions of PEV2 and PEV3 RNA genomes revealed approximately 1.0 kilobase (kb) RNA fragments located in the 5' regions of the two genomes. 3' RACE analysis determined that the size of the RNA fragments were 958 nucleotides (nt) for PEV2 and 968 nt for PEV3...

Peptic ulcer disease is generated by the activation of NF-kappaB activity. A recent clinical study reported a significant increase in NF-kappaB2 gene expression in 79 samples of peptic ulcer patients compared to the control group. Moreover, the deacetylated chitin could alter the translocation of NF-kappaB p65 to the nucleus. Considering this, our work aims to explore the effect of chitin extracted from the shell of blue swimming crabs ( Portunus pelagicus Linn.) towards NF-kappaB p65 levels in ethanol-induced gastric ulcerative Wistar rats...

Information on RNA localisation is essential for understanding physiological and pathological processes, such as gene expression, cell reprogramming, host-pathogen interactions, and signalling pathways involving RNA transactions at the level of membrane-less or membrane-bounded organelles and extracellular vesicles. In many cases, it is important to assess the topology of RNA localisation, i.e., to distinguish the transcripts encapsulated within an organelle of interest from those merely attached to its surface...

In recent years, there has been an increase in infectious diseases in marine mammals, including brucellosis, infections of morbillivirus, herpesvirus, and poxvirus. Several serological diagnostic methods, including enzyme-linked immunosorbent assays, immunofluorescence assays (ELISA), and western blotting, have been used to detect antibodies against pathogens in marine mammals. However, options for commercial secondary antibodies used to detect antibodies in marine mammals are limited; therefore, the use of proteins A, G, or chimeric protein AG may provide a suitable alternative...

Ribosomal RNA (rRNA) processing and maturation are fundamentally important for ribosome biogenesis, but the mechanisms in archaea, the third form of life, remains largely elusive. This study aimed to investigate the rRNA maturation process in Methanococcus maripaludis , a representative archaeon lacking known 3'-5' exonucleases. Through cleavage site identification and enzymatic assays, the splicing endonuclease EndA was determined to process the bulge-helix-bulge (BHB) motifs in 16S and 23S rRNA precursors...

Small RNAs (sRNAs) within 15-30 nt such as miRNA, tsRNA, srRNA with 3'-OH have been identified. However, whether these sRNAs are the major 15-30 nt sRNAs is still unknown. Here we show about 90% mammalian sRNAs within 15-30 nt end with 2',3'-cyclic phosphate (3'-cP). TANT-seq was developed to simultaneously profile sRNAs with 3'-cP (sRNA-cPs) and sRNA-OHs, and huge amount of sRNA-cPs were detected. Surprisingly, sRNA-cPs and sRNA-OHs usually have distinct sequences. The data from TANT-seq were validated by a novel method termed TE-qPCR, and Northern blot...

Culex pipiens, the northern house mosquito, is a major vector of West Nile virus. To survive the severe winter, adult mosquitoes enter a diapause programme. Extended lifespan and an increase in lipid storage are key indicators of diapause. Post-translational modifications to histone proteins impact the expression of genes and have been linked to the lifespan and energy utilisation of numerous insects. Here, we investigated the potential contribution of epigenetic alterations in initiating diapause in this mosquito species...