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In order to isolate the differentially expressed genes in the primordium stage of Pleurotus ostreatus, the SSH cDNA library was constructed using the cDNA from dikaryotic mycelium stage as a driver and the cDNA from primordium stage as a tester. There were 423 significantly differently expressed clones among 2055 positive clones after three times of reverse Northern blot differential screening. After the repeated sequences being removed, 46 genes were identified which were putatively involved in cell rescue and defense, energy metabolism, transcription and protein regulation, membrane proteins, and signal transduction; 18 genes encoding hypothetical proteins with unknown function; 5 genes without any homology. PoALDH1 and its full-length cDNA sequence were cloned using the Aldehyde dehydrogenase EST isolated from the library. The amino acid sequence of PoALDH1 contains conservative glutamic acid and cysteine residues active sites of aldehyde dehydrogenase family. When exposed to different concentrations of sodium chloride, the mycelium growth was inhibited and the expression level of PoALDH1 was significantly higher than that of the control one, which indicated that PoALDH1 may have the ability to relieve salt stress. The results of this study will provide useful information for isolating growth and development related genes of P. ostreatus.

Iron deficiency chlorosis is a significant yield-limiting problem in apple production, and screening or breeding for iron deficiency tolerant rootstocks is one of the best solutions. To increase the efficiency of selection for iron deficiency tolerant hybrids, a reverse northern blot-based dot-blot macroarray protocol was established in this paper. Using this macroarray, the expression levels of MxHA7, MxFIT1, MxIRT1, MxCS1 and MxRD3 in young roots of 141 interspecific hybrids between Malus xiaojinensis and M. baccata were analyzed on the third day after low iron (4 μM) treatment. Assisted by these gene expression markers, 14 iron deficiency tolerant lines were preliminarily selected out of the 141 hybrids. Then, 12 (85.7 %) preliminarily selections were confirmed by the phenotypes validation on the iron deficiency chlorosis index. These results indicate that our dot-blot macroarray is suitable for gene expression marker-assisted breeding for low iron stress tolerant apple rootstocks.

Sesuvium portulacastrum, a promising facultative halophyte species, has a remarkable ability to survival at severe salt of ~1000 mM NaCl. However, genes encoding proteins involved in the process of salt tolerance in S. portulacastrum are limited. In this study, suppression subtractive hybridization together with reverse northern blot analysis and RT-PCR was used to identify up-regulated genes in roots after treatment with seawater for 2–24 h. We identified a total of 148 unigenes, and most of these genes were predominantly associated with ‘signal transduction and transcription’, ‘stress/defense response’, ‘transport’ and ‘metabolism’. Comparative analysis between physiological response and differentially expressed genes highlighted that sodium (Na+) transport, compartmentation, reactive oxygen species (ROS) scavenging, and sugar-metabolizing regulation play important roles in salt tolerance of S. portulacastrum, whereas inhibition of root elongation induced by high salinity may be related to lignin deposition and disruption of calcium homeostasis.

Monoclonal antibodies (MAbs) are important tools for the study of proteins′ function and structure. But there has been no report on the preparation of MAbs against human KIAA0100 protein up to date. Here, first, we generated the mouse MAb against human KIAA0100 protein using purified recombinant 6×Histidinc (6×His)-tagged human KIAA0100 protein segment (1557–2234) as an antigen; then, the mRNA expression of human KIAA0100 gene was detected in U937 cells using Northern blot analysis. The results showed that the mouse MAb against human KIAA0100 protein could sensitively recognize the human KIAA0100 protein using Western blot analysis and immunocytochemistry analysis. Besides, Western blot analysis revealed that human KIAA0100 gene possibly encoded two different protein products (254 kDa and

N6-methyladenosine (m6A) is the most prevalent internal modification of eukaryotic messenger RNA (mRNA). The total amount of m6A can be detected by several methods, such as dot blot analysis using specific m6A antibodies and quantitative liquid chromatography-tandem mass spectrometry (LC-MS/MS) ( Fu et al., 2014 ; Shen et al., 2016 ). Here we describe the method for fast detection of total m6A levels in mRNA by dot blot analysis using a specific m6A antibody.

Small noncoding RNAs are essential for gene expression at transcriptional and posttranscriptional levels. Northern blot is the most used method for small RNA detection in tissues. Here we present an improved protocol for the Northern blot-based small RNA detection from plant tissues by using biotin-labeled probes. MicroRNAs and small interfering RNAs derived from Arabidopsis and Oryza sativa, respectively, have been detected with this methodology. Results suggest that this method is sensitive and efficient enough to detect small RNAs from plant tissues by using as low as 5 μg of total RNA. Furthermore, biotin-labeled probes are safer and easier to store for long term than radiolabeled probes.

Significant secretion of citrate from root apex of rice bean (Vigna umbellata) is delayed by several hours under aluminium (Al) stress. However, the molecular basis of regulation of VuMATE1, a gene encoding an Al-activated citrate transporter, remains unclear. In this study, we used suppression subtractive hybridization together with reverse northern blot analysis and qRT-PCR to identify genes with altered transcript levels in the root apex after treatment with low (5 μm) or high (25 μm) concentration of AlCl3 for a short time (4 h). We found that in addition to VuMATE1, 393 genes showed an early response to Al. Among functionally annotated genes, those related to ‘metabolism and energy’, ‘signal transduction and transcription’ and ‘transport’ was predominantly up-regulated, whereas those associated with ‘protein translation, processing and degradation’ was predominantly down-regulated. Comparative analysis of transcriptional profiles highlighted candidate genes associated with citrate secretion and revealed several new aspects of the molecular processes underlying Al toxicity and tolerance. Based on the data, it is proposed that metabolic changes represent adaptive mechanisms to Al stress, whereas inhibition of both cell elongation and cell division underlies Al-induced root growth inhibition.

Differential expressed genes in taxol producing Nodulisporium sylviforme HDF-68 at different periods of fermentation were analyzed by mRNA differential display. A total of 38 fragments were found expressed differentially during taxol synthesis phase. Using reverse Northern blot analysis, the hybridization signals of 16 fragments were consistent with the results of differential-display reverse transcription-PCR (DDRT-PCR). Sequencing results showed that nucleic acid sequences of TBR3, TBR4 and TBR7 were extremely similar to those of human, and the genes of cytochrome oxidase subunit I in TBR11 and Neurospora crassa mitochondrial were similar. No other functions could be identified by homolog analysis of the rest of expressed sequence tag (EST) sequences. In total, 16 open reading frames (ORFs) were found in these 16 ESTs, and seven of them have homologous proteins in the NCBI protein database. However, only the functions of TBR10 and TBR11 homolog were known; others were hypothetical proteins. Furthermore, the results also showed that the limitation of information about amplification product in 3′-UTR region could be overcome through a single random primer, instead of the anchor primer to combine with random primer in DDTR-PCR. This study provides valuable information for identifying functionally related genes in taxol-producing endophytic fungi and clues on further defining the taxol biosynthetic pathway.

Blotting techniques are among the most common approaches used in a molecular biology laboratory. These techniques, Southern, northern, and immunoblotting, are applicable to a variety of macromolecules including DNA, RNA, and protein, respectively. Each of the techniques are dependent on the ability to resolve the individual macromolecules in a size-dependant manner, transfer the molecules to a solid support, and finally use a defined probe to detect the specific molecule of interest. The utilization of the blotting technology over the last 30 years has been instrumental to the elucidation of many fundamental biological processes. The continued use of blotting technology holds promise for even greater discovery over the next 30 years. © 2016 by John Wiley & Sons, Inc. Keywords: Southern; Northern; Western; probe; hybridization; antibody; membrane; blotting; protein blot; DNA blot

Malignant gliomas are the most common and lethal intracranial tumors; differentiation therapy is a promising candidate for their treatment. In order to reveal the mechanisms related to glioma differentiation, after confirming that differentiation was induced by sodium phenylbutyrate in SHG-44 human glioma cells, RNA arbitrary primer differential display was used to screen differentially expressed genes. One gene was found to be upregulated by differential display, and this was also confirmed by reverse northern blot and quantitative real-time PCR analysis. After it was cloned and sequenced, the 505-bp fragment was identified as the MIBP1 (c-myc intron-binding protein 1) gene, also named Hivep2/MBP-2/Schnurri-2. Quantitative real-time PCR analysis of 30 human tissue samples revealed that the expression of MIBP1 tended to decrease with increasing WHO grade and was significantly depressed in the high malignancy gliomas group (WHO grade IV). We cloned and sequenced the MIBP1 gene, which was accepted by GenBank as number DQ231041. Finally, transfection of MIBP1 in a reverse transcription vector into glioma cells inhibited cell growth, induced differentiation, and blocked the cell cycle. Here, we identify and describe the structure and function of a differentiation-related gene, human MIBP1, in human glioma.

Roses are one of the most important cut flowers which are available in more color and sizes throughout the year. Senescence is a programmed event responding to external (environmental) and internal (developmental) signals; it requires de novo gene expression and protein synthesis, and is controlled in a highly coordinated manner. In order to know about molecular mechanisms and complex nets involved in aging regulating pathways, a cDNA-amplified fragment length polymorphism (cDNA-AFLP) analysis was performed to identify and isolate senescence-associated genes (SAGs) in rose petals of two cultivars ‘Black magic’ and ‘Marroussia’ with different longevities. The transcription profiles of plants at different developmental stages (flowering and senescent) were compared. About 1,600 cDNA fragments, ranging in size from 250 to 1,200 bp, were reproducibly detected. This allowed the identification of 22 differentially expressed cDNAs corresponding to genes belonging to different functional categories related to senescence, signal transduction pathway, carbohydrate metabolism, energy and other catabolic events such as protein, lipid and cell wall degradation. SAGs-encoded proteins are likely to participate in response to the two isolated fragments; RhCG (Rosa hybrida CG) and RhAA (Rosa hybrida AA) are more similar to putative protein associated to the senescence of Pisum sativum and Picea abies, respectively. There was no information for two fragments in the database. Reverse Northern blot demonstrated the credibility of the result of cDNA-AFLP. Study of expression pattern of the fragments showed an increase of expression level in almost all of them.

Repetitive genetic elements have had an unprecedented success populating phylogenetically diverse species making them a common feature of most genomes. Hundreds of thousands of copies of active and non-functional transposable elements representing different classes and families can reside within and outside of host genes. In addition to creating structural variations in genomic DNA, some of these loci are expressed to contribute to the continuing amplification cycle. Transposable elements, specifically Long Interspersed Element-1 (LINE-1) produce a spectrum of RNAs, some of which are important for their mobilization, while others are processed forms of LINE-1 transcription that may or may not play relevant functions. Additionally, many LINE-1 sequences integrated into cellular genes are included into cellular transcripts creating substantial background when L1-related RNA expression is detected by some conventional methods. This chapter provides an in-depth description of the complexity of L1-generated mRNAs as well as sources of cellular transcripts containing L1 sequences. It also highlights the strengths and weaknesses of conventional methods used to detect LINE-1 expression.

The gene differential expression of Phytophthora sojae during early stage (0 to 7 h) of sporangium formation was studied by differential display reverse transcription polymerase chain reaction (DDRT-PCR) in the present study. Thirty out of 78 primer combinations were selected and 90 stable differential bands were obtained. Thirty-nine positive differential fragments were found in the experiments by using the reverse northern blot, in which 36 sequences of differential fragments were obtained after sequencing. Homology search and analysis were tested by BLASTX in the P. sojae genome database. The results showed that 23 fragments had significant similarities with proteins in P. sojaegenome database, 15 of which matched with the known functional proteins involved in metabolism, cellular processes, and signal transduction processes. Data mining and bioinformatics analysis on 4 fragments which redundancy was greater than 5 showed that sequence AR15-258 had a very high expression level in Phytophthora mycelium, which may be related to mycelium infection or hunger; AR10-263 and GR1-304 were only found in Phytophthora, which might be unique expression in this genus. The analysis and research on important regulatory genes controlling sporangium formation of P. sojae was a foundation for studying of key genes controlling growth and development of P. sojae. Key words: Phytophthora sojae, sporangium formation, mRNA differential display, differential fragments.

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.

Mycorrhizal fungi promote the growth and development of plants, including medicinal plants. The mechanisms by which this growth promotion occurs are of theoretical interest and practical importance to agriculture. Here, an endophytic fungus (AR-18) was isolated from roots of the orchid Anoectochilus roxburghii growing in the wild, and identified as Epulorhiza sp. Tissue-cultured seedlings of A. roxburghii were inoculated with AR-18 and co-cultured for 60 d. Endotrophic mycorrhiza formed and the growth of A. roxburghii was markedly promoted by the fungus. To identify genes in A. roxburghii that were differentially expressed during the symbiosis with AR-18, we used the differential display reverse transcription polymerase chain reaction (DDRT-PCR) method to compare the transcriptomes between seedlings inoculated with the fungus and control seedlings. We amplified 52 DDRT-PCR bands using 15 primer combinations of three anchor primers and five arbitrary primers, and nine bands were re-amplified by double primers. Reverse Northern blot analyses were used to further screen the bands. Five clones were up-regulated in the symbiotic interaction, including genes encoding a uracil phosphoribosyltransferase (UPRTs; EC 2.4.2.9) and a hypothetical protein. One gene encoding an amino acid transmembrane transporter was down-regulated, and one gene encoding a tRNA-Lys (trnK) and a maturase K (matK) pseudogene were expressed only in the inoculated seedlings. The possible roles of the above genes, especially the UPRTs and matK genes, are discussed in relation to the fungal interaction. This study is the first of its type in A. roxburghii.

5-hydroxymethylcytosine (5hmC) is considered as a novel DNA modification and plays an important role in cancer, stem cells, and developmental diseases. In this study, we demonstrated the existence of RNA 5hmC modification in mouse brain RNA by using a dot blot analysis method. Our data indicated that 5hmC modification in RNA samples was less than that in DNA samples. Further, we optimized the conditions for 5hmC detection in RNA samples such as DNase treatment, denature reagents, denature time, sample air-dry time, and the cross-linking time between RNA and membrane. Our results demonstrated that DNase treatment and denature reagents were two important factors that affected the 5hmC detection in RNA samples. By using the optimal conditions for RNA 5hmC detection, we found that the brainstem, the hippocampus, and the cerebellum had high levels of 5hmC modification and 5mC modification in RNA. Finally, we found that RNA 5hmC modification decreased in MPTP-induced Parkinson's disease model in mice. These suggest that 5hmC modification in RNA might play an important regulative role on protein or microRNA expression in these brain tissues. Because DNA 5hmC modification plays an important role in neural differentiation and development as well as neurological diseases, the significance of 5hmC modification in RNA in different neurological diseases needs further investigation. In summary, our study demonstrated for the first time the abundance of 5hmC modification in brain RNA by using a dot blot analysis method and proved that dot blot analysis is a useful method for 5hmC detection in RNA samples.

To analyze the differentially expressed genes between resistant and susceptible wheat near-isogenic lines infected by Puccinia striiformis Westend. f. sp. tritici, a subtractive library containing about 1300 clones was constructed using suppression subtractive hybridization (SSH) in which the cDNA from resistant Yr4/6 × Taichung 29 seedlings inoculated with race CY26 was used as the tester, and the corresponding cDNA from susceptible Taichung 29 as the driver. Six hundred clones from the library were analyzed with reverse Northern blot. The positive clones were further tested by Northern blotting analysis. Twelve clones were verified and showed significant difference. By means of sequencing and BlastX analysis, six function-known differentially expressed sequences were detected, and their putative products were leucine-rich repeat protein, catalase, thioredoxin H-type, RNA binding protein, ascorbate peroxidase, and heat shock protein, respectively. Among them, leucine-rich repeat protein belongs to signal transduction protein, and others belong to defense response protein.

This protocol describes how to perform northern blot analyses to detect microRNAs and their precursors with single-nucleotide resolution, which is crucial for analyzing individual length variants and for evaluating relative quantities of unique microRNAs in cells. Northern blot analysis consists of resolving RNAs by gel electrophoresis, followed by transferring and fixing to nylon membranes as well as detecting by hybridization with radioactive probes. Earlier efforts to improve this method focused mainly on altering the sensitivity of short RNA detection. We have enhanced the resolution of the northern blot technique by optimizing the electrophoresis step. We have also investigated other steps of the procedure with the goal of enhancing the resolution of RNAs; herein, we present several recommendations to do so. Our protocol is applicable to analyses of all kinds of endogenous and exogenous RNAs, falling within length ranges of 20–30 and 50–70 nt, corresponding to microRNA and pre-microRNA lengths, respectively.

This study was designed to construct and identify the subtracted cDNA library in peripheral blood cells of BALB/c mice and tracheal-bronchial epithelial cells of Wistar rats following exposure to radon inhalation. Two groups of the animals were exposed in a radon chamber at an accumulative dose of 100 WLM, while control animals were housed in a room at a background dose of 1 WLM. To construct a subtracted cDNA library enriched with differentially expressed genes, the SMART technique and suppression subtractive hybridization (SSH) assay were performed. The obtained forward and reverse cDNA fragments were directly inserted into a pMD-18 vector and transformed into Escherichia coli JM109. In total, 593 white bacterial clones were selected from both forward- and reverse-subtracted libraries. Among them, 81 clones were chosen for their differential expressions based on reverse Northern blot. Portions of these cDNA clones were also verified by a quantitative real-time polymerase chain reaction (PCR). The screening resulted in 14 upregulative and 8 downregulative known function/annotation genes, which were revealed to be functionally related to cell proliferation, cell oxidative and DNA damage, apoptosis, and tumor promotion. Access numbers were obtained from the GenBank for 11 unknown expressed sequence tags (EST). Analysis of biological roles of these cDNA fragments may provide further insight into mechanisms underlying adverse molecular events induced by high-dose radon exposure.

In the reproductive organs of maize (Zea mays L.), the changes in transcription that occur during meiosis at early stage of water deficit were characterized. We used oligo microarray analysis, which included 57452 transcripts representing more than 30 000 genes, and combined this with reverse Northern blot analysis. After 1 d stress, 1 809 transcripts were differentially expressed (by 2-fold or greater) with 34 % of them being upregulated in the tassels, while in the ears 861 transcripts were differentially expressed with 41 % being upregulated. Of these, 33 transcripts were upregulated in both organs, including those involved in protective functions, reactive oxygen species detoxification, nitrogen metabolism and gibberellin metabolism. In contrast, the transcripts involved in polyamine and sugar metabolism were downregulated.

Glucose-6-phosphate dehydrogenase (G6PD) deficiency is a common inherited disorder worldwide including southern China. We developed and validated a reverse dot blot (RDB) assay for the rapid and simultaneous genotyping of six mutations (c.95A>G, c.871G>A, c.1004C>T, c.1024C>T, c.1376G>T and c.1388G>A), that were common mutations in the Chinese G6PD deficiency population, and one polymorphism (c.1311C>T). Reliable genotyping of wild-type and mutant genomic DNA samples was achieved by means of a test strip onto which allele-specific oligonucleotide probe lines are fixed in parallel. This method involves a multiplex PCR amplification of three fragments in the G6PD target sequence and a manual hybridization/detection protocol. The entire procedure starting from blood sampling to the identification of mutations requires less than 6 h. The diagnostic reliability of this reverse dot blot assay was evaluated on 207 pre-typed samples by using direct DNA sequence analysis in a blind study. The reverse dot blot typing was in complete concordance with the reference method. The reverse dot blot assay was proved to be a simple, rapid, highly accurate, and cost-effective method to identify common G6PD mutations in Chinese population.

Identifying parasitism genes encoding proteins secreted from nematodes is the key to understanding the molecular basis of nematode parasitism to insects. In this paper, a cDNA with two introns and three exons encoding a cysteine protease inhibitor was identified by screening a cDNA subtractive library constructed from the nematode, Steinernema carpocapsae, induced by Galleria mellonella hemolymph. The full-length cDNA contains an open reading frame encoding a 139-amino acid protein, designated Sc-cys, with a 19-residue signal peptide. The mature protein was predicted to have a molecular weight of 12,531.59 Da, a pI of 9.44, one disulfide bond, and three conserved domains believed to be important for the inhibition of cysteine proteases. In Basic Local Alignment and Search Tool analyses, the putative protein precursor displayed 26-42% identities to a multitude of cystatins or cystatin-like proteins. Phylogenetic analysis suggested the novel cystatin is likely a new member of the family 2 cystatins. Reverse northern blot, semi-quantitative reverse transcriptase polymerase chain reaction (RT-PCR), and real-time RT-PCR analyses showed that the expression level of Sc-cys was upregulated substantially after induction by insect hemolymph. The specific analysis of genes encoding secretory proteins is providing a profile of putative parasitism genes expressed in S. carpocapsae throughout the parasitic cycle.

Early metastasis and a poor five-year survival make lung cancer the leading cause of cancer related deaths worldwide. The clinical profile of lung cancer patients in India differs from the West as they present earlier, with squamous cell carcinoma being the commonest histological type. We compared gene expression profiles in primary lung squamous cell carcinoma (LSCC) and matched normal lung tissues in Asian Indians. Using suppression subtractive hybridization, two subtracted cDNA libraries containing differentially expressed genes in the tumors were constructed. Differential expression was confirmed by reverse Northern blot analysis. DNA of confirmed clones was sequenced and subjected to GenBank Blast searches. RNA expression levels were then analyzed by Northern blotting and validated by semiquantitative RT-PCR (in 10 cases of NSCLC). Seventeen differentially expressed gene cDNA fragments of LSCC were analyzed. The differentially expressed genes included those associated with cellular metabolism, cell-cycle, -structure, -adhesion, transcription, proliferation, apoptosis and signal transduction. The study provided first evidence that KIAA0767, a Death Inducing Protein, a novel p53 independent target of E2F1, and Geminin, an inhibitor of DNA replication are differentially expressed in LSCC. Identification of the differentially expressed genes in lung cancer in this study may serve as better molecular markers for early diagnosis and identifying novel intervention sites for anticancer therapy.

CMS-D8 and its restorer were developed by introducing the cytoplasm and nuclear gene Rf (2) from the wild diploid Gossypium trilobum (D8) into the cultivated tetraploid Upland cotton (Gossypium hirsutum). No information is available on how the Rf (2) gene interacts with CMS-associated genes and how CMS-D8 cytoplasm affects nuclear gene expression. The objective of this study was to identify differentially expressed genes in anther tissues between the non-restoring fertile maintainer ARK8518 (rf(2) rf(2)) and its isogenic heterozygous D8 restorer line, ARK8518R (Rf(2) rf(2)) with D8 cytoplasm, by mRNA differential display (DD). Out of more than 3,000 DDRT-PCR bands amplified by 31 primer combinations from 12 anchor primers and 8 arbitrary decamer primers, approximately 100 bands were identified as being qualitatively differentially displayed. A total of 38 cDNA fragments including 12 preferentially expressed cDNA bands in anther were isolated, cloned and sequenced. Reverse northern blot analysis showed that only 4 genes, including genes encoding a Cys-3-His zinc finger protein and aminopeptidase, were up-regulated, while 22 genes, including genes for phosphoribosylanthranilate transferase (PAT), starch synthase (SS), 4-coumarate-CoA ligase, electron transporter, calnexin, arginine decarboxylase, and polyubiquitin, were down-regulated in the heterozygous restorer ARK8518R. The down-regulation of SS explains the lack of starch accumulation in sterile rf(2) pollen grains in the heterozygous restored plants. The molecular mechanism of CMS and its restoration, specifically the possible roles of SS and PAT genes in relation to restoration of Rf(2) to CMS-D8, are discussed. This investigation represents the first account of such an analysis in cotton.

Rice sheath blight, caused by Rhizoctonia solani is one of the major diseases of rice. The pathogen infects rice plants directly through stomata or using lobate appressoria and hyphal masses called infection cushions. The infection structures were normally found at 36 h post-inoculation. During infection, the pathogenesis-related genes, PR1b and PBZ1 were induced in rice plants. To identify rice genes induced early in the defense response, suppression subtractive hybridization (SSH) was used to generate a cDNA library enriched for transcripts differentially expressed during infection by R. solani. After differential screening by membrane-based hybridization and subsequent confirmation by reverse Northern blot analysis, selected clones were sequenced. Fifty unique cDNA clones were found and assigned to five different functional categories. Most of the genes were not previously identified as being induced in response to pathogens. We examined expression of 100 rice genes induced by infection with Magnaporthe grisea, Xanthomonas oryzae pv. oryze (Xoo) and X. oryzae pv. oryzicola (Xooc). Twenty-five of them were found to be differentially expressed after the sheath blight infection, suggesting overlap of defense responses to different fungal and bacterial pathogens infection.

Tapping panel dryness (TPD) occurrence in high latex yielding rubber tree (Hevea brasiliensis) is characterized by the partial or complete cessation of latex flow upon tapping leading to severe loss in natural rubber production around the world. The goal of this study was to identify genes whose mRNA transcript levels are differentially regulated in rubber tree during the onset of TPD. To isolate TPD responsive genes, two cDNA libraries (forward and reverse) from total RNA isolated from latex of healthy and TPD trees were constructed using suppression subtractive hybridization (SSH) method. In total, 1,079 EST clones were obtained from two cDNA libraries and screened by reverse Northern blot analysis. Screening results revealed that about 352 clones were differentially regulated and they were selected for sequencing. Based on the nucleotide sequence data, the putative functions of cDNA clones were predicted by BLASTX/BLASTN analysis. Among these, 64 were genes whose function had been previously identified while the remaining clones were genes with either unknown protein function or insignificant similarity to other protein/DNA/EST sequences in existing databases. RT-PCR analysis was carried out to validate the up-regulated genes from both the libraries. Among them, two genes were strongly down-regulated in TPD trees. The level of mRNA transcripts of these two genes was further examined by conventional Northern and RT-PCR analysis. Results indicated that the expression level of two genes was significantly lower in TPD trees compared to healthy trees. Many TPD associated genes were also up-regulated in TPD trees suggesting that they may be involved in triggering programmed cell death (PCD) during the onset of TPD syndrome. The results presented here demonstrate that SSH technique provides a powerful complementary approach for the identification of TPD related genes from rubber tree.

To identify components of the plant stress signal transduction cascade and response mechanisms, we screened plant genes using reverse Northern blot analysis, and chose the ethylene responsive element binding protein 1 (StEREBP1) for further characterization. To investigate its biological function in the potato, we performed Northern blot analysis and observed enhanced levels of transcription in response to several environmental stresses including low temperature. In vivo targeting experiments using a green fluorescent protein (GFP) reporter indicated that StEREBP1 localized to the nucleus of onion epidermal cells. StEREBP1 was found to bind to GCC and DRE/CRT cis-elements and both microarray and RT-PCR analyses indicated that overexpression of StEREBP1 induced expression of several GCC box-containing stress response genes. In addition, overexpression of StEREBP1 enhanced tolerance to cold and salt stress in transgenic potato plants. The results of this study suggest that StEREBP1 is a functional transcription factor that may be involved in abiotic stress responses in plants.

Combining the procedures of immunoprecipitation and immunoblotting can help to overcome some of the limitations of each separate procedure. Immunoblotting can identify immunoprecipitated proteins more specifically and with higher sensitivity than nonspecific protein stains or autoradiography. Immunoprecipitation can enrich proteins of interest to improve sensitivity for detection when compared with immunoblotting of whole cell extracts. Recently, immunoprecipitation-blotting helped to characterize a new autoantibody, anti-p155, and to test for the presence of the autoantibody in patient sera to study its clinical associations. The procedure for immunoprecipitation-blotting, with specific reference to this autoantibody test ("reverse" immunoprecpitation-blotting), is reported here in detail.

Successful detection of very small RNAs (tiny RNA, ~14 nt in length) by Northern blotting is dependent on improved Northern blot protocols that combine chemical crosslinking of RNA with 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC) to positively charged membranes, the use of native polyacrylamide gels, and the development of highly sensitive and specific probes modified with locked nucleic acids (LNA). In this protocol, we show that Northern blot detection of tiny RNAs with 5'-digoxigenin-labeled DNA/LNA mixmer probes is a highly sensitive and specific method and, in our hands, more sensitive than using a corresponding DNA/LNA mixmer probe with a 5'-(32)P-end label.

We prepared total RNA from the Gram-positive soil bacterium Bacillus subtilis by different RNA extraction procedures to compare their suitability for Northern blot detection of tiny RNAs (~14-mers) or RNAs of intermediate size (100-200 nt) in terms of signal quality, intensity, and reproducibility. Our analysis included two hot phenol methods and two TRIzol extraction procedures. We found that signal intensity/detection sensitivity makes the key difference. Total RNAs prepared by the hot phenol method comprise the length spectrum from tRNAs to large ribosomal RNAs. Larger RNAs are less abundant in TRIzol preparations which instead enrich for RNAs of tRNA size and smaller. Thus, hot phenol methods are the choice for the detection of intermediate-sized and longer RNAs, whereas TRIzol extraction procedures are more suited for the detection of tiny RNAs.

The present study was attempted to identify transcriptionally regulated genes of the normal neurocytes responsive to iron availability. Postnatal rat hippocampus cells were primarily cultured either under the iron-loaded or depleted conditions. These cultured cells were applied for the generation of subtracted complementary DNA libraries by the suppression subtraction hybridization (SSH) and for the subsequent identification of differentially expressed transcripts by reverse Northern blot. The differentially expressed genes were chosen to perform sequencing, and then some of them were performed by Northern blot analysis for observation of their expression in the hippocampus of rats with the different iron status. The results indicated that five unique transcripts were strong candidates for differential expression in cellular iron repletion, one of them is a novel sequence (GenBank No. AF 433878), while 26 unique transcripts were strong candidates for differential expression in cellular iron deprivation, one of them is a novel sequence (GenBank No. AY 912101). The revealed known genes responsive to iron availability were previously unknown to respond to iron availability, or have not been determined in the brain, have not even been currently determined in their physiological and biological functions. Interestingly, the proteins encoded by most of the known genes are either directly pointed to or indirectly associated with the molecules that play important, even key roles in cellular signal transduction and the cell cycle. These findings lead to the important suggestion that the cellular responses to iron availability involve extensive transcriptional regulation and cellular signal transduction. Therefore, iron may serve as a signal, which directly and/or indirectly regulates or modulates cell functions.

Oligochitosan has a variety of biological activities. To understand its mechanism, DDRT-PCR, reverse Northern blot and quantitative relative RT-PCR were used to identify and isolate genes whose transcription were altered in cultured Nicotiana tabacum (var. Samsun NN) plants that were treated with oligochitosan. Three genes whose mRNA levels significantly changed in response to oligochitosan were isolated and identified. One gene is up-regulated, and two genes are down-regulated. These genes encode a DNAJ heat shock N-terminal domain-containing protein, a histone H1 gene and a hypothetical protein, whose function is unknown. The results suggest that the usefulness of mRNA differential display technique for the detection of plant metabolic pathways affected by oligochitosan.

Arbuscular mycorrhizal fungi are able to alleviate the stress for plants caused by heavy metal contamination of soil. To analyze the molecular response of arbuscular mycorrhizal fungi to these pollutants, a subtractive cDNA library was constructed using RNA from Glomus intraradices extraradical hyphae of a root organ culture treated with a mixture of Cd, Zn, and Cu. Screening by reverse Northern blot analysis indicated that, among 308 clones, 17% correspond to genes up-regulated by heavy metals. Sequence analysis of part of the clones resulted, amongst others, in the identification of six genes putatively coding for glutathione S-transferases belonging to two different classes of these enzymes. Expression analyses indicated that the genes are differentially expressed during fungal development and that their RNA accumulation dramatically increases in extraradical hyphae grown in a heavy metal-containing solution.

Several studies have demonstrated that the Rhizobium nifA gene is an activator of nitrogen fixation acting in nodule bacteria. To understand the effects of the Sinorhizobium meliloti nifA gene on Alfalfa, the cDNA-AFLP technique was employed to study the changes in gene expression in nifA mutant nodules. Among the approximately 3,000 transcript-derived fragments, 37 had differential expression levels. These expression levels were subsequently confirmed by reverse Northern blot and RT-polymerase chain reaction. Sequence analyses revealed that 21 cDNA fragments corresponded to genes involved in signal communication, protein degradation, nutrient metabolism, cell growth and development.

In order to unravel the biochemical pathways and understand the molecular mechanisms involved in leaf senescence, suppression subtractive hybridization (SSH) was used to generate a cDNA library enriched for transcripts differentially expressed in developmental senescence cotyledons of upland cotton. After differential screening by membrane-based hybridization and subsequent confirmation by reverse Northern blot analysis, selected 678 clones were sequenced and analyzed. Sequencing of these cDNA fragments reveals that 216 of expressed sequence tags (ESTs) represented unique genes. Of these 216 cDNAs, 151 clones (69.9%) show significant homologies to previously known genes, while the remaining 65 do not match any known sequences. 151 unique ESTs are assigned to twelve different categories based on their putative functions generated by BLAST analysis. These SAG-encoded proteins are likely to participate in macromolecule degradation, nutrient recycling, detoxification of oxidative metabolites, and signaling and regulatory events. The expression pattern of selection of genes was confirmed using northern hybridization. Northern hybridization confirmed several distinct patterns, from expression at a very early stage to the terminal phase of the senescence syndrome. Clones encoding proteases and proteins involved in macromolecule degradation and gluconeogenesis, as well as stress-related genes, are up regulated in senescence cotyledons.

Differentially expressed genes in chickpea, ( Cicer arietinum L.) during root infection by Fusarium oxysporum f sp. ciceri Race1, were identified using cDNA-RAPD and cDNA-AFLP approaches. The former employed decamer primers on cDNA template and revealed nine differentially expressed transcripts in the resistant-infected chickpea variety. Among the 2000 transcript-derived fragments (TDFs) screened by cDNA-AFLP, 273 were differentially expressed in chickpea roots during Fusarium infection. Only 13.65% of the TDFs were differentially regulated during pathogen challenge, while the other 86% were expressed non-differentially during the process of pathogen infection in chickpea roots. Nineteen TDFs, which expressed differentially in the resistant-infected chickpea variety were cloned and sequenced. Two of these TDFs were similar to transcription factors like WRKY proteins and 14-3-3 proteins, while three TDFs represented the NBS-LRR-type gene sequences. Two TDFs had sequence identity to genes known to have function in defense. The RAPD TDF CaFRi60 showed sequence identity to gamma-glutamyl-cysteine synthetase. Among the TDFs examined by cDNA-AFLP, 19 were confirmed by Reverse Northern blot to be differentially expressed. The data confirms the effectiveness of the cDNA-AFLP technique in detecting differentially expressed genes during pathogenesis.

MicroRNAs (miRNAs) are an important class of endogenously derived, small approximately 22 nucleotide noncoding regulatory RNAs that have recently become implicated in development, cell regulation and cancers of various tissues. Here we report a nonisotopic Northern analysis method for miRNA detection using 3'-digoxigenin (DIG)-labeled RNA oligo probes. Northern blot analysis was performed using miRNA or total RNA fractions extracted from human leukemic cell lines, and blots were hybridized with either 32P- or DIG-labeled RNA probe for miR-181, miR-155 or miR-16. A labeled probe for U6 small nuclear RNA served as an internal control. The use of DIG-labeled RNA probes was equally sensitive compared to 32P-labeled probes in detecting miRNA quantities as low as 50 ng. The ability to use nonisotopic methods and yet obtain sensitive and reliable results offers an advantage to investigators who prefer to avoid isotopes.

Representational difference analysis of cDNA (cDNA-RDA) was used for a comparison of the global transcript level of tumor of the larynx and the corresponding normal epithelial tissue toward the end of detecting differentially expressed genes. Overall, some 130 gene fragments were identified. By sequence analysis and homology comparison, they could be put into several groups related to (potential) functions. Apart from genes whose overexpression was most likely a result of tumor growth or dedifferentiation of epithelial tissue, a lot of genes were isolated that play major roles in signal transduction pathways or apoptosis or act as oncogenes or tumor suppressor genes, in addition to new, entirely unknown genes. Moreover, some cDNAs of known genes were identified that derived from unconventional splicing activity or other transcript modifications. All identified fragments were arrayed on solid support and used for reverse Northern blot analyses. The use of preselected RDA fragments as targets in array-based profiling experiments circumvents many of the problems encountered when dealing with large clone libraries.

We describe a technique designed to assess the optimal dilution of primary and secondary antibodies, to be used in Western blot, dot blot, the multi-antigen print immunoassay (MAPIA) and immunohistochemistry assays. The method that we call "line blot" is not an alternative but a practical, complementary tool for the above techniques that assures definitive results are obtained from single assays, so there is no need to repeat the assay. As with most immunoenzymatic assays, the line blot assay is very sensitive, allowing the detection of absolute amounts of antigen as low as 2.5 ng in the 0.5 cm-long segment line (see Results), depending on the strength of the secondary, enzyme-labelled antibody.

Somatic embryogenesis (SE) is the developmental reprogramming of somatic cells toward the embryogenesis pathway and is a notable illustration of cell totipotency. To identify genes involved in SE, subtractive polymerase chain reaction (PCR) was performed to generate transcripts highly enriched for SE-related genes, using cDNA prepared from a mixture of embryogenic callus and pre-globular somatic embryos, as the tester, and cDNA from non-embryogenic callus, as the driver. After differential screening and subsequent confirmation by reverse Northern blot analysis, a total of 671 differentially expressed cDNA fragments were identified, and 242 uni-genes significantly up-regulated during cotton SE were recovered, as confirmed by Northern blot and reverse-transcription PCR analysis of representative cases, including most previously published SE-related genes in plants. In total, more than half had not been identified previously as SE-related genes, including dominant crucial genes involved in transcription, post-transcription, and transportation, and about one-third had not been reported previously to GenBank or were expected to be unknown, or newly identified genes. We used cDNA arrays to further investigate the expression patterns of these genes in differentiating gradient culture, ranging from pro-embryogenic masses to somatic embryos at every stage. The cDNA collection is composed of a broad repertoire of SE genes which is an important resource for understanding the genetic interactions underlying SE signaling and regulation. Our results suggested that a complicated and concerted mechanism involving multiple cellular pathways is responsible for cotton SE. This report represents a systematic and comprehensive analysis of genes involved in the process of somatic embryogenesis.

DNA methyltransferases (DNMTs) and 5-methyl-CpG-binding domain proteins (MBDs) are involved in the acquisition of parent-specific epigenetic modifications in human male and female germ cells. Reverse Northern blot analyses demonstrated sex-specific differences in mRNA expression for the maintenance DNMT1 and the de novo DNMT3A in developing testis and ovary. In fetal testis DNMT1 and DNMT3A expression peaked in mitotically arrested spermatogonia around 21 weeks gestation. In fetal ovary transcriptional upregulation of DNMT1 and DNMT3A occurred during a very brief period at 16 weeks gestation, when the oocytes proceeded through meiotic prophase. Fetal gonads showed several fold higher DNMT3A expression levels than fetal brain and adult tissues. The most abundant DNMT3A isoform in fetal testis and ovary was DNMT3A2, whereas in all other analyzed tissues DNMT3A1 predominated. The catalytically inactive DNMT3A3 isoform was also present at relatively high levels in developing gonads and may perform a regulatory function(s). In both male and female fetal gonads expression of genes for MBD2 and MBD4, which may be implicated in chromatin remodeling of methylated genomic DNA sequences, was tightly linked to DNMT expression. We propose that the sex-specific time windows for concomitant upregulation of DNMT1, DNMT3A, MBD2, and MBD4 are associated with prenatal remethylation of the human male and female germ line.

In 1994, the bacterial parasite Mycoplasma gallisepticum expanded its host range and swept through populations of a novel host — eastern US populations of the house finch ( Carpodacus mexicanus ). This epizootic caused a dramatic decline in finch population numbers, has been shown to have caused strong selection on house finch morphology, and presumably caused evolutionary change at the molecular level as finches evolved enhanced resistance. As a first step toward identifying finch genes that respond to infection by Mycoplasma and which may have experienced natural selection by this parasite, we used suppression subtractive hybridization (SSH) and cDNA macroarray approaches to identify differentially expressed genes regulated by the Mycoplasma parasite. Two subtractive cDNA libraries consisting of 16 512 clones were developed from spleen using an experimentally uninfected bird as the ‘tester’ and an infected bird as ‘driver’, and vice versa. Two hundred and twenty cDNA clones corresponding 34 genes with known vertebrate homologues and a large number of novel transcripts were found to be qualitatively up- or down-regulated genes by high-density filter hybridization. These gene expression changes were further confirmed by a high throughout reverse Northern blot approach and in specific cases by targeted Northern analysis. BLAST searches show that heat shock protein (HSP) 90, MHC II-associated invariant chain (CD74), T-cell immunoglobulin mucin 1 (TIM1), as well as numerous novel expressed genes not found in the databases were up- or down-regulated by the host in response to this parasite. Our results and macroarray resources provide a foundation for molecular co-evolutionary studies of the Mycoplasma parasite and its recently colonized avian host.