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The transcription of mammalian genomes exhibits an intriguing complexity and numerous novel RNA molecules have been identified. Viruses with large DNA genomes, especially herpesviruses, generate many different RNA species, including long non-coding RNAs (lncRNAs). Dense viral genomes can generate multigenic transcripts in addition to commonly observed antisense transcripts. It is essential to study the biological roles of these transcripts aside from the protein-coding counterparts. Multiple antisense transcripts from the open reading frame (ORF) 63-64 locus in murine gammaherpesvirus 68 (MHV68) were detected by northern blotting. Expression analysis by quantitative reverse transcription PCR (qRT-PCR) did not detect different isoforms. Several alternative splicing isoforms exist during lytic replication; however, they are not detected during latency. To identify the roles of these new transcripts, qRT-PCR may not be enough and should be supported by an alternative method such as northern blotting. A more detailed transcriptional map of the locus of interest is useful to design experimental strategies and perform functional studies, especially when working with gene-dense viral genomes.

Northern blotting is a common technique in RNA biology, allowing to detect and quantify RNAs of interest following separation by gel electrophoresis, transfer to a membrane, and hybridization of specific anti-complementary labelled probes. In this chapter, we describe our protocol for efficient RNA extraction from yeast, separation on agarose gel, and capillary transfer to a membrane. We provide two different methods for strand-specific detection of several types of RNAs using oligonucleotide probes, the first using radioactive 32 P-labelled probes, the second based on nonradioactive digoxigenin-labelled probes., (© 2025. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Advances in sequencing technology have led to the discovery of diverse types of regulatory RNAs. Differential transcript levels regulate cellular processes and influence disease severity. Identifying these variations through reliable methods is crucial for understanding the regulatory roles and disease mechanisms of regulatory RNAs. Northern blotting, which is considered the gold standard for differential expression analysis, poses challenges due to various limitations associated with RNA quality and integrity, radioactivity exposure, and associated reagents and expenses. In this protocol, we employ a biotin-based northern blotting (BiNoB) approach that is both convenient and inexpensive, eliminating the need for specialized settings as required with radioactivity-based northern blotting. We comprehensively target various RNA types, making this technique a versatile tool for RNA detection. Additionally, we conduct a comparison between 3'-end labeled probes that were labeled in-house and 5'-end labeled probes that were obtained commercially. Remarkably, our results reveal relatively higher sensitivity with 3'-end labeled probes. Furthermore, we demonstrated that the use of an in-house buffer offered comparable sensitivity to a commercially available buffer, providing another cost-effective alternative. We also aimed to determine the minimum quantity of total RNA required to detect small non-coding RNAs such as tRNA fragments. Whereas previous studies reported the use of 5-10 µg total RNA for tRNA fragment detection, our findings revealed that as little as 1 µg total RNA is sufficient to detect small RNAs like tRNAs and their fragments. This concentration may vary depending on the expression levels of the specific RNAs being detected. © 2024 Wiley Periodicals LLC. Basic Protocol: Biotin-based northern blotting., (© 2024 Wiley Periodicals LLC.)

Northern blotting (NB), a gold standard for RNA detection, has lost its charm due to its hands-on nature, need for good quality RNA, and radioactivity. With the emergence of the field of microRNAs (miRNAs), the necessity for sensitive and quantitative NBs has again emerged. Here, we developed highly sensitive yet non-radiolabeled, fast, economical NB, and liquid hybridization (LH) assays without radioactivity or specialized reagents like locked nucleic acid (LNA)- or digoxigenin-labeled probes for mRNAs/small RNAs, especially miRNAs using biotinylated probes. An improvised means of hybridizing oligo probes along with efficient transfer, cross-linking, and signal enhancement techniques was employed. Important caveats of each assay were elaborated upon, especially issues related to probe biotinylation, use of exonuclease, and bioimagers not reported earlier. We demonstrate that, while the NBs were sensitive for mRNAs and small RNAs, our LH protocol could efficiently detect these and miRNAs using less than 10–100 times the total amount of RNA, a sensitivity comparable to radiolabeled probes. Compared to NBs, LH was a faster, more sensitive, and specific approach for mRNA/small RNA/miRNA detection. A comparison of present work with six seminal studies is presented along with detailed protocols for easy reproducibility. Overall, our study provides effective platforms to study large and small RNAs in a sensitive, efficient, and cost-effective manner.

Investigation of RNAs with a length less than 30 nucleotides, in particular, microRNAs and other extremely short noncoding RNAs, is often associated with difficulties in detecting them not only in vivo, but also in vitro. One of these molecules is bacterial pRNA (product RNA) synthesized by RNA polymerase on 6S RNA as a template. The classical method for identifying pRNA is Northern blot hybridization with complementary DNA probes containing various labels and modified nucleosides. However, this method is not suitable for searching for new pRNAs whose sequences are unknown. It is also quite expensive and time-consuming. We have proposed a new simple way to determine the fact of pRNA synthesis on the template of 6S RNA that we called "mirror-like" Northern blotting. It does not require gel electrophoresis, and the pRNA, newly formed during transcription, act as a probe itself due to incorporated digoxigenin (DIG) residues. This approach will allow fast screening of new 6S RNAs or their mutant variants for their ability to serve as templates for pRNA synthesis and the estimation of its efficacy. [ABSTRACT FROM AUTHOR]

Background: Virulence genes and the expression of resistance mechanisms undoubtedly play a role in the successful spread of the pandemic clone Escherichia coli ST131. Porin down-regulation is a chromosomal mechanism associated with antibiotic resistance. Translation of porin proteins can be impacted by modifications in mRNA half-life and the interaction among small RNAs (sRNAs), the porin transcript and the sRNA chaperone Hfq. Modifications in the translatability of porin proteins could impact the fitness and therefore the success of E. coli ST131 isolates in the presence of antibiotic.Objectives: To identify differences in the translatability of OmpC and OmpF porins for different STs of E. coli by comparing steady-state RNA levels, mRNA half-life, regulatory sRNA expression and protein production.Methods: RNA expression was evaluated using real-time RT-PCR and OmpC mRNA half-life by northern blotting. OmpC, OmpF and Hfq protein levels were evaluated by immunoblotting.Results: Differences between ST131 and non-ST131 isolates included: (i) the level of OmpC RNA and protein produced with mRNA expression higher for ST131 but OmpC protein levels lower compared with non-ST131 isolates; (ii) OmpC mRNA half-life (21-30 min for ST131 isolates compared with <2-23 min for non-ST131 isolates); and (iii) levels of the sRNA MicC (2- to 120-fold for ST131 isolates compared with -4- to 70-fold for non-ST131 isolates).Conclusions: Mechanisms involved in the translatability of porin proteins differed among different STs of E. coli. These differences could provide a selective advantage to ST131 E. coli when confronted with an antibiotic-rich environment. [ABSTRACT FROM AUTHOR]

This study examined the molecular mechanisms underlying the roles of the microRNAs miR-18a and miR-25 in the progression of human liver cancer. Liver cancer biopsies obtained from early-stage liver cancer patients were examined by qRT-PCR and Northern blotting to examine the expression of miR-18a and miR-25. Both microRNAs were overexpressed in mouse primary hepatocytes following transfection of the cells with vectors encoding the microRNAs. An analysis of biopsy samples from liver cancer patients indicated that both miR-18a and miR-25 were overexpressed during the early stages of liver cancer. Further, qRT-PCR and Northern blotting confirmed that both of these microRNAs play crucial roles in the progression of liver cancer. Our findings clearly indicate that miR-18a and miR-25 can be used as prognostic biomarkers for early-stage liver cancer. Hence, miR-18a and miR-25 may have value as prognostic indicators and may facilitate the development of novel therapeutics for liver cancer. [ABSTRACT FROM AUTHOR]

RNA Interference (RNAi), which works against invading nucleic acids or modulates the expression of endogenous genes, is a natural eukaryotic regulating system, and it works by noncoding smaller RNA molecules. Plant-mediated gene silencing through RNAi can be used to develop plants with insect tolerance at transcriptional or post-transcriptional levels. In this study, we selected Myzus persicae’s acetylcholinesterase 1 gene (Ace 1) as a silencing target to develop transgenic Solanum lycopersicum L. plants’ resistance to aphids. An RNAi plasmid vector containing an artificial microRNA (amiRNA) sequence was engineered and successfully transformed into Jamila and Tomaland, two elite tomato cultivars. A northern blot analysis and PCR were carried out to check the efficacy of Agrobacterium-mediated transformation in T0 transgenic plants. The quantitative PCR data showed a substantial downregulation of the Ace 1 gene in aphids fed in clip cages on T1 transgenic plants. Furthermore, there was a substantial drop in aphid colonies that were fed on T1 transgenic plants of both the cultivars. These findings strongly suggest that transgenic plants that express amiRNA could be an important tool for engineering plants resistant to aphids and possibly for the prevention of viral disease in other plant-infested pests.

Cultivation of human melanocytes requires several growth factors for cell proliferation. For example, basic fibroblast growth factor (bFGF) is an essential growth agent for melanocyte proliferation in vitro and has been proposed to be an autocrine growth factor in human melanoma cells. Studies using either anti-bFGF antibodies or antisense oligonucleotides partially inhibited the proliferation of human melanoma cells. However, one group was unable to detect bFGF RNA transcripts in human melanoma cells using a human complementary DNA probe. These contradictory results prompted us to investigate the bFGF gene expression in human primary melanocytes and metastatic melanoma cells using Southern, Northern, and Western blot analyses. No gross rearrangements in the bFGF gene were detected in the genomic DNA. Although high levels of bFGF RNA transcripts were detected in melanocytes, no bFGF protein was detected using Western blot analysis. In contrast, melanoma cells expressed much lower levels of bFGF RNA transcripts, and cells from three of four cell strains synthesized the multiple isoforms of bFGF protein. In one of the melanoma cell strains, no bFGF protein was detected using Western blot analysis. Although three of four melanoma cell strains expressed bFGF protein, this molecule does not appear to function as an autocrine growth factor, and expression of the bFGF protein was not a consistent alteration in all melanoma cell strains.

In this chapter, we describe in vivo methods for the analysis of interactions between an sRNA and its target mRNA in B. subtilis. All these methods have been either established or significantly improved in our group and successfully employed to characterize a number of sRNA/target mRNA systems in Bacillus subtilis. Whereas in Chap. 8, we describe a combination of in vitro methods, e.g., EMSA and RNA secondary structure probing, we focus here on the investigation of RNA-RNA interactions in vivo using compatible plasmids or chromosomal insertions and deletions, the elucidation of the mechanisms of action of regulatory sRNAs employing transcriptional and translational reporter gene fusions, as well as the determination of expression profiles, half-lives of sRNA and mRNA, and their intracellular concentrations, and, finally, the investigation of RNA chaperones that promote the sRNA/mRNA interaction. For an in-depth analysis of sRNA-mRNA interactions in B. subtilis, a combination of in vivo and in vitro methods should be applied., (© 2024. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

In the study, we successful constructed a library from the ovine testis using next-generation sequencing technology, and identified 219 novel miRNAs by bioinformatics. Two of the novel miRNAs (ovis_aries_testis-m0052_5p and ovis_aries_testis-m0165_5p), which were expressed in the sheep testis and ovary, and were confirmed by real-time PCR and northern blotting. Ovis_aries_testis-m0052_5p was 23 nucleotides in length, was located on chromosome 15, and had 100% similarity to mmu-miR-34c-5p, hsa-miR-34c-5p, gga-miR-34c-5p, and cfa-miR-34c. Ovis_aries_testis-m0165_5p was 21 nucleotides in length, located on chromosome 5, and had 100% similarity to mmu-miR-145a-5p, hsa-miR-145-5p, ssc-miR-145-5p, and bta-miR-145. The pre-miRNAs for Ovis_aries_testis-m0052_5p and Ovis_aries_testis-m0165_5p were 75 and 81 nucleotides in length, and both had a standard hairpin stem-loop structure. From the consistency of the sequence and structure, we speculated that ovis_aries_testis-m0052_5p had a function similar to hsa-miR-34c-5p, mmumiR- 34c-5p, and ovis_aries_testis-m0165_5p had a function similar to hsa-miR-145-5p, which were involved in the fine regulation of cell survival, spermatozoon generate, breeding activities. Therefore, we defined the identified miRNAs as oar-miR-34c-5p and oar-miR-145-5p. The results will enrich the miRNA database, and provide the basis for research into the regulatory mechanisms of miRNA in relation to breeding activities. [ABSTRACT FROM AUTHOR]

In bacteria, small non-coding RNAs (sRNAs) are critical regulators of cellular adaptation to changes in metabolism, physiology, or the external environment. In the last decade, more than 2000 of sRNA families have been reported in the Rfam database and have been shown to exert various regulatory functions in bacterial transcription and translation. However, little is known about sRNAs and their functions in Mesorhizobium. Here, we predicted putative sRNAs in the intergenic regions (IGRs) of M. huakuii 7653R by genome-wide comparisons with four related Mesorhizobial strains. The expression and transcribed regions of candidate sRNAs were analyzed using a set of high-throughput RNA deep sequencing data. In all, 39 candidate sRNAs were found, with 5 located in the symbiotic megaplasmids and 34 in the chromosome of M. huakuii 7653R. Of these, 24 were annotated as functional sRNAs in the Rfam database and 15 were recognized as putative novel sRNAs. The expression of nine selected sRNAs was confirmed by Northern blotting, and most of the nine selected sRNAs were highly expressed in 28 dpi nodules and under symbiosis-mimicking conditions. For those putative novel sRNAs, functional categorizations of their target genes were performed by analyzing the enriched GO terms. In addition, MH_s15 was shown to be an abundant and conserved sRNA.

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. This allows establishing which RNAs can, in principle, engage in local molecular interactions and which are prevented from interacting by membranes or other physical barriers. The most widely used techniques interrogating RNA localisation topology are based on the treatment of isolated organelles with RNases with subsequent identification of the surviving transcripts by northern blotting, qRT-PCR, or RNA-seq. However, this approach produces incoherent results and many false positives. Here, we describe Controlled Level of Contamination coupled to deep sequencing (CoLoC-seq), a more refined subcellular transcriptomics approach that overcomes these pitfalls. CoLoC-seq starts by the purification of organelles of interest. They are then either left intact or lysed and subjected to a gradient of RNase concentrations to produce unique RNA degradation dynamics profiles, which can be monitored by northern blotting or RNA-seq. Through straightforward mathematical modelling, CoLoC-seq distinguishes true membrane-enveloped transcripts from degradable and non-degradable contaminants of any abundance. The method has been implemented in the mitochondria of HEK293 cells, where it outperformed alternative subcellular transcriptomics approaches. It is applicable to other membrane-bounded organelles, e.g., plastids, single-membrane organelles of the vesicular system, extracellular vesicles, or viral particles. Key features • Tested on human mitochondria; potentially applicable to cell cultures, non-model organisms, extracellular vesicles, enveloped viruses, tissues; does not require genetic manipulations or highly pure organelles. • In the case of human cells, the required amount of starting material is ~2,500 cm 2 of 80% confluent cells (or ~3 × 10 8 HEK293 cells). • CoLoC-seq implements a special RNA-seq strategy to selectively capture intact transcripts, which requires RNases generating 5'-hydroxyl and 2'/3'-phosphate termini (e.g., RNase A, RNase I). • Relies on nonlinear regression software with customisable exponential functions., Competing Interests: Competing interestsThere are no conflicts of interest or competing interests., (©Copyright : © 2023 The Authors; This is an open access article under the CC BY-NC license.)

Black pepper (Piper nigrum L.), member of the family Piperaceae is indigenous to India and is one of the most widely used spices in the world. In this paper we report the results of our attempts to identify a set of genes differentially expressed in the leaves of Piper nigrum, which could facilitate targeted engineering of this valuable crop. A PCR-based Suppression Subtractive Hybridization (SSH) technique was used to generate a leaf-specific subtracted cDNA library of Piper nigrum. A tester population of leaf cDNA was subtracted with a root derived driver cDNA. The efficiency of subtraction was confirmed by PCR analysis using the housekeeping gene actin. On sequence analysis, almost 30% of the clones showed homology to metallothionein type-2 gene. The predominance of metallothionein transcripts in the leaf was further confirmed using Real-Time PCR analyses and Northern blot. The possible role of metallothionein type-2 homologues in the leaf is discussed along with the feasibility of using SSH technique for identification of more number of tissue-specific genes from Piper nigrum.

The article describes in detail methodical procedures for isolation of small RNA molecules, their electrophoresis, northern blotting and finally detection by hybridization with radioactively labeled riboprobes. The presented protocols were successfully introduced and used in our laboratory.

Ganoderma lingzhi is a kind of precious medicinal mushroom. MicroRNAs (miRNAs) play an important role in the regulation of a broad range of biological processes. In this study, we first report on the miRNAs of the G. lingzhi sporocarp. In total, 132 known miRNAs and 34 putative candidate (PC) miRNAs were identified for the first time in G. lingzhi sporocarp. Through target gene prediction, 111 of 132 known miRNAs had targeted genes, 58 of them had more than one, and most predicted targeted genes might be regulated by more than one miRNA; functional enrichment by KEGG analysis demonstrated that target genes related to cancer and signal transduction occupied the largest proportion. A total of seven miRNAs (four known miRNAs and three PC miRNAs) that had high abundance were selected for expression confirmation by quantitative real-time PCR (qRT-PCR) and northern blotting. This is the first comprehensive study of the miRNA composition in G. lingzhi , which resulted in the addition of a significant number of novel miRNA sequences to miRBase and laid the foundation for further understanding of the function of miRNAs in the regulation of G. lingzhi. [ABSTRACT FROM AUTHOR]

Fluoroquinolone (FQNL) antibiotics have been used widely in horses because of their broad-spectrum bactericidal activity and relative safety, however, their use is not without risk. Tendonitis and spontaneous tendon rupture have been reported in people during or following therapy with FQNLs. To evaluate the potential damage of enrofloxacin (ENRO) on the equine superficial digital flexor tendon (SOFT), an equine cell culture system as an in vitro model of equine tendon injury and repair was developed. The effects of ENRO on tendon cell cultures established from equine SOFT were studied. The data thus collected demonstrated that ENRO inhibited cell proliferation, induced morphological changes and altered proteoglycan synthesis in equine tendon cell cultures. Interestingly, these effects were more pronounced in juvenile tendon cells as compared to adult horses, It. is hypothesized that morphological changes and inhibition of cell proliferation is a result of impaired production of proteoglycans and other glycoproteins in the extracellular matrix of ENRO-treated tendon cells.

The effects of atorvastatin on the expression of the hepatic HMG-CoA reductase and LDL receptor genes were investigated in rats. Like the other statins, atorvastatin increased the rate of degradation and presumably cycling of the hepatic LDL receptor. In atorvastatin-treated rats, the half-life of the receptor was decreased by over 60%. Hepatic HMG-CoA reductase mRNA levels were increased about 3-fold by feeding a diet containing 0.04% atorvastatin while reductase protein levels were increased by as much as 700-fold. Apparent HMG-CoA reductase activity was not increased as much as protein levels. Washing experiments revealed that atorvastatin is more difficult to remove from microsomes than lovastatin. The results support the conclusion that the potent hypocholesterolemic action of atorvastatin involves decreased hepatic VLDL production due to effective inhibition of in vivo cholesterol biosynthesis resulting from diminished recovery of HMG-CoA reductase activity following drug treatment.—Ness, G. C., C. M. Chambers, and D. Lopez. Atorvastatin action involves diminished recovery of hepatic HMG-CoA reductase activity. J. Lipid Res. 1998. 39: 75–84.

Background: Recently identified porcine circovirus-like virus P1 has the smallest DNA viral genome. In this study, we identified the viral genes and their corresponding mRNA transcripts. Results: The RNAs of P1, synthesized in porcine kidney cells, were examined with northern blotting and PCR analyses. Eight virus-specific RNAs were detected. Four mRNAs (open reading frames (ORFs) 1, 2, 4, and 5) are encoded by the viral (−) strand and four (ORFs 3, 6, 7, and 8) are encoded by the viral (+) strand. All proteins encoded by the ORFs of the P1 virus are less than 50 amino acids in length, except that encoded by ORF1 (113 amino acids). Conclusions: We show a very complex viral transcription pattern in P1-infected cells. [ABSTRACT FROM AUTHOR]

Adding ethidium bromide (EtBr) at low concentrations to RNA samples before running formaldehyde-agarose gels affords the advantages of checking RNA integrity and evaluating the quality of size-separation at any time during electrophoresis or immediately after either electrophoresis or blotted the separated RNA onto the membrane without significantly compromising mobility, transfer, or hybridization. In this study, we systematically examined the factors that affect the sensitivity of RNA prestaining by heating RNA samples that include EtBr before electrophoresis under different denaturation conditions. We also examined the efficiency of the hybridization of EtBr-prestained RNA with heterologous DNA probes. The results showed that the fluorescent intensity of EtBr-prestained RNA was affected not only by the EtBr concentration as previously reported but also by the RNA amount, denaturation time, and denaturation temperature. Prior staining of RNA with 40 μg/mL EtBr significantly decreased the efficiency of Northern blot hybridization with heterologous DNA probes. We propose that to best combine staining sensitivity and the efficiency of Northern blot hybridization with heterologous DNA probes, the concentration of EtBr used to prestain RNA should not exceed 30 μg/mL. The efficiency of the hybridization of EtBr-prestained RNA was affected not only by factors that affect staining sensitivity but also by the type of probe used. [ABSTRACT FROM AUTHOR]

Based on the significant differences in the resistance to Sclerotinia sclerotiorum between two Brassica napus cultivars, ZhongR888 and Zhongyou821, near isogenic lines (NILs) were established. The resistance differences between Zhongyou821 and BC progeny (the fifth generation from a cross between Zhongyou821 and ZhongR888 with Zhongyou821 as successive backcross parent) was displayed by cDNA-AFLP and differential transcripts-derived fragments (TDFs). The full-length cDNA of 1,707 bp with 1,323 bp open reading frame (ORF, open box), named Rsk was identified by RT-RACE from the resistance related fragment 40-2 in Zhongyou821. It encoded a protein of 440 amino acid residues, which contained a putative extracellular domain, a transmembrane domain, and an intracellular domain. Analysis of the deduced amino acid sequence of Rsk revealed that it had high homology to Arabidopsis thaliana kinase involved in protein binding, and had a conserved region of LRR-RI, indicating it might be a member of leucine-rich repeats, ribonuclease inhibitor-like subfamily. DNA sequences of 1,685 bp and 1,703 bp without an intron were also identified from Zhongyou821 and BC respectively. Expression analysis showed that Rsk might play a role in disease resistance pathways. [ABSTRACT FROM AUTHOR]

Abstract: Pathogenic isolates of Fusarium oxysporum applied on a non-host plant species, as soil-borne non-pathogenic isolates, are able to protect this plant against pathogenic strains inducing wilts. Several modes of action contribute to the biocontrol activity of these protective strains; however the genetic basis of the biocontrol mechanisms is far from being understood. The aim of this study was to identify genes involved in biocontrol activity of F. oxysporum using an original model made of Fom24, a strain protective on tomato and its mutant rev157 which has lost its protective capacity. A Rapid Subtractive Hybridization (RaSH) approach was chosen to identify genes up-regulated in the protective or in the non-protective interaction when germinated conidia of either Fom24 or rev157 are confronted to tomato cell cultures. A total of 86 up-regulated sequences were generated, 42 and 44 from the protective and the non-protective interaction respectively. Homology searches led to identification of both plant and fungal genes that were grouped according to their putative functions. Among plant genes, those involved in plant response to stresses were the most abundant. Expression profiles of genes homolog to a basic endochitinase, a ferredoxine-NADP(H) reductase (FNR), an ATP synthase and the RPM1-interacting protein 4 (RIN4) were confirmed by Northern blotting. A large proportion of fungal sequences were encoding genes of unknown function; among other, those involved in response to oxidative stress and a gene putatively encoding an enolase are the most promising to further study their potential role in the protective interaction between F. oxysporum and tomato. [Copyright &y& Elsevier]

polipoprotein E (apoE) is predominantly a lipid transport protein involved in membrane repair and synaptic plasticity in brain. Its genotype is associated with late-onset sporadic and familial Alzheimer's disease (AD). Its expression increases when any injury takes place in the nervous system and in age-related disorders including AD. However, it is not clearly understood how the level of apoE changes in the normal aging brain. Therefore, we have analyzed the effect of age on apoE protein and mRNA level in the cerebral cortex of young, adult, and old male AKR strain mice. Western, northern, and RT-PCR results reveal that the level of apoE protein and mRNA is higher in young as compared to adult and then remains unchanged in old mouse cerebral cortex. This indicates that during normal aging, apoE level is maintained in old similar to adult and suggests involvement of other factors in maintaining apoE-mediated brain functions in old. [ABSTRACT FROM AUTHOR]

MicroRNAs (miRNAs) are a new family of small RNA molecules found in plants and animals. We developed a comprehensive strategy for identifying new miRNA homologues by mining the repository of available citrus expressed sequence tags (ESTs). By adopting a range of filtering criteria, we identified a total of 38 potential miRNAs - nine, five, nine and 15 miRNAs in Citrus trifoliata (ctr-miRNAs), C. clementina (ccl-miRNAs), C. reticulata (crt-miRNAs) and C. sinensis (csi-miRNAs), respectively - from more than 430,000 EST sequences in citrus. Using the potential miRNA sequences, we conducted a further BLAST search of the mRNA database and found six potential target genes in these citrus species. Eight miRNAs were selected in order to verify their existence in citrus using Northern blotting, and the functions of several miRNAs in miRNA-mediated gene regulation are experimentally suggested. It appears that all these miRNAs regulate expression of their target genes by cleavage, which is the most common situation in gene regulation mediated by plant miRNAs. Our achievement in identifying new miRNAs in citrus provides a powerful incentive for further studies on the important roles of these miRNAs. [ABSTRACT FROM AUTHOR]

The molecular mechanisms of the endogenous circadian clocks that allow most animals to adapt to environmental cycles have recently been uncovered. The draft genome of the ascidian, Ciona intestinalis, a model animal that is close to vertebrates, has been described. However, the C. intestinalis genome lacks the canonical clock genes such as Per, Bmal and Clock that are shared by vertebrates and insects. Here, we found the circadian rhythms at the physiological and molecular levels. The oxygen consumption rate was lower during the light phase and higher during the dark phase during a day, and the rhythm highly damped and continued under constant darkness. From the microarray analysis, the 396 spots (1.8% of the total; corresponding to 388 clones) were extracted as candidates for circadian expression. We confirmed the circadian expression of several candidate genes by northern blotting. Furthermore, three of four rhythmic expressed genes showed phase-shifts to prolonged light period. However, most of known clock genes did not oscillate. These data suggest that C. intestinalis have a unique molecular circadian clock and the daily environmental change is not such a strong effect for sea squirt in its evolution when compared to vertebrates and insects. [ABSTRACT FROM PUBLISHER]

In mammals, 10 isoforms of fatty acid-binding protein (FABP) are expressed in various tissues. To understand the role of multiple FABP isoforms, we have quantitatively examined the transcript levels of individual FABP isoforms in each of various tissues by Northern blotting using synthesized RNAs corresponding to the mRNA of each isoform as external standards. As a result, absolute transcript levels of individual FABP isoforms expressed in each tissue were successfully determined. The 10 FABP isoforms were classified into three categories: (1) isoforms FABP7 and 12 were not markedly expressed in any tissue examined; (2) isoforms showing certain transcript levels in multiple tissues; and (3) isoforms FABP6, 8, and 9, expressed at certain levels in one particular tissue. Based on the expression profiles of the isoforms, individual tissues were also classified into three groups: (1) tissues in which high-level expression of FABP isoforms was not observed, (2) tissues in which multiple FABP isoforms were expressed at certain levels, and (3) tissues in which a single FABP isoform was dominantly expressed at a certain level. These results give a better understanding of the meaning of the presence of multiple FABP isoforms in mammals. [ABSTRACT FROM AUTHOR]

MicroRNAs (miRNAs) are a class of non-protein-coding small RNAs. Considering the conservation of many miRNA genes in different plant genomes, the identification of miRNAs from non-model organisms is both practicable and instrumental in addressing miRNA-guided gene regulation. Citrus is an important staple fruit tree, and publicly available expressed sequence tag (EST) database for citrus are increasing. However, until now, little has been known about miRNA in citrus. In this study, 27 known miRNAs from Arabidopsis were searched against citrus EST databases for miRNA precursors, of which 13 searched precursor sequences could form fold-back structures similar with those of Arabidopsis. The ubiquitous expression of those 13 citrus microRNAs and other 13 potential citrus miRNAs could be detected in citrus leaf, young shoot, flower, fruit and root by northern blotting, and some of them showed differential expression in different tissues. Based on the fact that miRNAs exhibit perfect or nearly perfect complementarity with their target sequences, a total of 41 potential targets were identified for 15 citrus miRNAs. The majority of the targets are transcription factors that play important roles in citrus development, including leaf, shoot, and root development. Additionally, some other target genes appear to play roles in diverse physiological processes. Four target genes have been experimentally verified by detection of the miRNA-mediated mRNA cleavage in Poncirus trifoliate. Overall, this study in the identification and characterization of miRNAs in citrus can initiate further study on citrus miRNA regulation mechanisms, and it can help us to know more about the important roles of miRNAs in citrus. [ABSTRACT FROM AUTHOR]

The receptor for advanced glycation end-products (RAGE) is thought to be expressed ubiquitously as various protein isoforms. Our objective was to use Northern blotting, immunoblotting, and sensitivity to N-glycanase digestion to survey RAGE isoforms expressed in cell lines and mouse tissues in order to obtain a more comprehensive view of the RAGE expressome. Pulmonary RAGE mRNA (1.4 kb) was smaller than cell-line and tissue RAGE mRNA (6 kb-10 kb). Three anti-RAGE antibodies that recognized three distinct RAGE epitopes were used for protein studies (N-16, H-300, and αES). Lung expressed three predominant protein isoforms with apparent molecular masses of 45.1, 52.6, and 57.4 kDa (N-16/H-300) and four isoforms at 25.0, 46.9, 52.5, and 54.2 kDa (αES). These isoforms were expressed exclusively in lung. Heart, ileum, and kidney expressed a 44.0-kDa isoform (N-16), whereas aorta and pancreas expressed a 53.3-kDa isoform (αES). Each of these isoforms were absent in tissue extracts prepared from RAGE−/− mice. Cell lines expressed a 70.0-kDa isoform, and a subset expressed a 30.0-kDa isoform (αES). Lung RAGE appeared to contain two N-linked glycans. Tissue and cell-line RAGE isoforms were completely insensitive to PNGase F digestion. Thus, numerous RAGE protein isoforms are detectable in tissues and cell lines. Canonical transmembrane and soluble RAGE appear to be expressed solely in lung (N-16/H-300). Non-pulmonary tissues and cell lines, regardless of the source tissue, both express distinct RAGE protein isoforms containing the N-terminal N-16 epitope or the αES RAGE epitope encoded by alternate exon 9, but lacking the H-300 epitope. [ABSTRACT FROM AUTHOR]

Intergeneric hybridization is an important strategy to introgress alien genes into common wheat for its improvement. But presence of crossability barrier mechanism regulated by Kr1 gene played a major destructive role for hybridization than other reported genes. In order to know the underlying molecular mechanism and to dissect out this barrier, cDNA-AFLP (Amplified Fragment Length Polymorphism) transcriptional analysis was carried out using chromosome 5B RIL (Recombinant Inbred Line) population. Totally 14 differentially expressed fragments for Kr1 gene were identified, cloned and sequenced. Further the expression was confirmed by northern blotting analysis. Sequence analysis of the resulted clones revealed several classes of putative genes, including stress responsive, signal transduction and flowering related genes. The probable relation between other genes, Vrn1 and Ph1, located along with Kr1 gene was discussed. [ABSTRACT FROM AUTHOR]

Microsporidia are a group of intracellular parasites characterized by highly reduced and compact genomes. The presence of a high gene density had several consequences for microsporidian genomes, including a high frequency of overlap between transcripts of adjacent genes. This phenomenon is apparently widespread in microsporidia, and strongly correlated with gene density. However, all analyses to date have focused on one or a few transcripts from many loci, so it is unclear how diverse the pool of transcripts at a given locus may be. To address this question, we characterized initiation and termination points from 62 transcripts in gene-dense regions in Antonospora locustae spores using both conventional and fluorescence-based RACE-PCR procedures. In parallel, we investigated the abundance and nature of transcripts along a 6 kb region surrounding the actin locus of A. locustae using northern blotting, RACE-PCR and previously characterised EST sequences. Overall, we confirmed previous suggestions that most transcripts in A. locustae spores overlap with the downstream gene, but that at the 5′ end untranslated regions are very short and overlap is rare . From fluorescence-based RACE-PCR we show that transcription of most genes (31 out of 34) initiates at a single position, whereas 35% of loci analyzed with 3′ RACE polyadenylate mRNA at several sites. Finally, we identified the presence of previously unsuspected and very large transcripts in A. locustae spores. Those transcripts were found to overlap up to four open reading frames in different strands, adding a novel layer of complexity in the mRNA transcription of this microsporidian species. [ABSTRACT FROM AUTHOR]

The article presents a study which analyzed signaling events related to cell cyle control in human ovarian cancer (OC) using primary culture and mRNA analysis. An introduction to related studies is offered. Details of the study's methodology including RNA isolation and Northern blot analysis, cRNA probe synthesis and ovarian cancer cell isolation and culture, among others, are described. Also discussed are findings of the study like the choice of medium for culturing human ovarian surface epithelial (OSE) and ovarian cancer cells.

The silk glands of Bombyx mon L. have been used as a model system in the production of large amount of silk proteins prior to cocoon spinning. The amount of silk proteins synthesized could be augmented by improving the nutritional status of silkworm larvae. The present study reports the changes in the silk gene expression by supplementing P-soyatose (hydrolyzed soy protein) during the fifth instar B. mon male larvae. Western blot analysis of the silk gland protein in the soy protein supplemented group demonstrated higher staining intensity of H and L chains. The rate of protein synthesis was increased in the male larva reared with P-soyatose in comparison to control group. This was indicated by high incorporation of 3H leucine in the posterior silk glands of supplemented larvae reared with P-soyatose. Northern blot analysis unequivocally prove that supplementation of soy protein increases the synthesis of silk fibroin, as it increases the levels of fibroin mRNA, which reflects fortification in the economic traits of B. mori. [ABSTRACT FROM AUTHOR]

Abstract: We studied metallothionein (MT) response in the manure worm Eisenia fetida after exposures to cadmium (Cd), zinc (Zn) or cadmium and zinc spiked media. MT was studied both at the protein level by Dot Immunobinding Assay, (DIA) and at the expression level by Northern blotting. Cd was highly accumulated by worms whereas Zn body concentration was regulated. In addition, Zn would limit Cd accumulation in worms exposed to low Cd concentrations (1 and 8 mg Cd kg−1 of dry soil). Exposure to a mixture of Cd and Zn at high concentrations increased cytosolic MT levels. This increase would allow worms to regulate body Zn concentrations and also to limit Cd toxicity. Cd exposures increased gene expression of Cd-binding MT isoform (MT 2A) whereas Zn did not. However, when both metals were at high concentrations in the exposure medium, this expression was further increased. Several hypotheses are proposed to explain the results and the best approach to estimate metal exposure of this earthworm species is given. Further experiments have now to be performed to evaluate the usefulness of these MT responses for field contaminated soils toxicity assessment using this earthworm species. [Copyright &y& Elsevier]

The nif genes of Clostridium beijerinckii NRRL B593 occupy a region of about 16 kilobases. Besides the two glnB-like genes, five other genes are interspersed between the nifNB and the nifVωgenes. An expression analysis of the nifgenes in nitrogen-fixing and non-nitrogen-fixing cells with probes generated from various regions of the nif cluster by northern blot analysis revealed the presence of four different transcripts in nitrogen-fixing cells. Two of these transcripts had the predicted sizes spanning from nifH to nifK and from nifVω to nifVα, respectively. The other two transcripts did not have the expected sizes, but they suggested the presence of two other polycistronic mRNAs consisting of nifE-nifNB and nirJ1-nirJ2-nirD-nirH, respectively. The absence of the nif and nif-associated mRNAs in RNA samples from non-nitrogen-fixing cells indicated that nif-associated genes are regulated in parallel to nif genes. [ABSTRACT FROM AUTHOR]

OBJECTIVE To inhibit the expression of the vascular endothelial growth factor (VEGF) by RNA interference, and to observe the effect in different cells line. METHODS Using the services of E-RNAi, we designed and constructed two kinds of shRNAs expression vectors which were aimed at the VEGF gene. These vectors were then transfected into HEK293, colon cancer HT29, Hela and HepG2 cells by Lipofectamine TM 2000. The level of VEGF mRNA was determined by RT-PCR and Northern blotting and the VEGF expression was examined by immunofluoresence staining. RESULTS The two kinds of VEGF specific shRNAs expression vectors were found to efficiently inhibit the expression of VEGF in HEK293 and HT29 cells by RT-PCR analysis, with inhibition rates of 72% and 42%, respectively; but the inhibition rates were reduced to 28% in Hela cells and 13% in HepG2 cells. Northern blotting showed that the inhibition rates of VEGF mRNA expression were 88% and 89% in HEK293 and HT29 cells, respectively. The inhibition rate of VEGF protein expression in HT29 cells was 73% based on immunofluoresence staining. CONCLUSION The expression of VEGF was inhibited by RNA interference, but differed with various cells lines, showing that RNA interference was cell-line dependent. [ABSTRACT FROM AUTHOR]

Significant progress has been made in unraveling the molecular biology of rice in the past two decades. Today, rice stands as a forerunner amongst the cereals in terms of details known on its genetics. Evidence show that salt tolerance in plants is a quantitative trait. Several traditional cultivars, landraces, and wild types of rice like Pokkali, CSR types, and Porteresia coarctata appear as promising materials for donation of requisite salt tolerance genes. A large number of quantitative trait loci (QTL) have been identified for salt tolerance in rice through generation of recombinant inbred lines and are being mapped using different types of DNA markers. Salt-tolerant transgenic rice plants have been produced using a host of different genes and transcript profiling by micro- and macroarray-based methods has opened the gates for the discovery of novel salt stress mechanisms in rice, and comparative genomics is turning out to be a critical input in this respect. In this paper, we present a comprehensive review of the genetic, molecular biology, and comparative genomics effort towards the generation of salt-tolerant rice. From the data on comprehensive transcript expression profiling of clones representing salt-stress-associated genes of rice, it is shown that transcriptional and translational machineries are important determinants in controlling salt stress response, and gene expression response in tolerant and susceptible rice plants differs mainly in quantitative terms. [ABSTRACT FROM AUTHOR]

Phospholipase D (PLD) and phosphatidylcholine (PC) were implicated in apoptosis and cancer. However, direct evidence on the role of PLD in the cause of apoptosis remains obscure. It was recently reported that apoptosis and necrosis could be induced in the cerebellum and brainstem after focal cerebral hypoxic-ischemic (HI) injury. It was found that apoptosis could be enhanced by farnesol inhibition of PLD signal transduction. Whereas it was shown that highly invasive cancer cell line depends on PLD activity for survival when deprived of serum growth factors. Based on these reports, it is postulated that apoptosis in the cerebellum and brainstem induced after focal cerebral HI treatment may be caused by faulty PLD expression. This is consistent with a report that PLD1 activity and mRNA levels were down-regulated during apoptosis. To test this hypothesis, Northern blotting was used to examine PLD2 mRNA expression after focal cerebral HI. The results show that both PLD2 mRNA 10.8 and 3.9 kb transcripts were significantly decreased by as much as 37% in the brainstem and cerebellum areas 3 h after HI compared to the control, concur with previous report of decreasing PLD activity after ischemia. These PLD2 transcripts, however, were not significantly different from the control 3 days after HI, indicating that the decrease in PLD2 transcription after HI maybe a transient phenomenon. This is the first report to show that the loss of membrane integrity resulting from deprivation of energy and growth factors after HI could cause decrease in PLD2 transcription that promotes apoptosis. The hypothetic role of PLD2 and the mechanism leading to apoptosis remains to be further elucidated. [ABSTRACT FROM AUTHOR]

Abstract: We studied the metallothionein (MT) response in cadmium-exposed worms (Eisenia fetida) both at the protein level by Dot Immunobinding Assay (DIA) with a polyclonal antibody raised against the most immunogenic part of this protein and at the expression level by Northern blotting using a specific probe. MT appeared as two close isoforms. DIA results clearly demonstrated significant differences in MT level of whole worm heat-treated supernatants between E. fetida exposed to Cd concentrations as low as 8 mg Cd kg−1 of dry soil compared to controls. Northern blotting analysis performed on whole bodies of worms revealed that a single exposure to 8 mg Cd kg−1 of dry soil for 1 day resulted in the production of MT mRNA. This response was maintained for exposure of at least 1 month. Clear differences of MT gene expression were also observed between worms exposed to different Cd concentrations (8, 80 or 800 mg Cd kg−1 of dry soil). Immunocytochemistry demonstrated that MT was located in the chloragogenous tissue surrounding the gut where metals are known to be accumulated. This work revealed that E. fetida MT is a sensitive and relevant biomarker of Cd exposure and especially when considering gene expression response. Further experiments have now to prove its usefulness in natural metal-contaminated soil toxicity assessments. [Copyright &y& Elsevier]