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At least 19 recordsLinked to original sources
PubMed1988

Comparison of dot-blot and Northern blot hybridizations in the determination of genetic relatedness of United States bluetongue virus serotypes.

Dot-blot and Northern blot hybridization methods to determine the genetic relatedness of United States bluetongue virus serotypes 2, 10, 11, 13, and 17 were compared. Both plasmid and insert DNA probes from cloned BTV-17 dsRNA segments 2, 5, 6, and 8 were hybridized to dsRNA from the BTV serotypes and epizootic hemorrhagic disease virus (EHDV). Stringencies of hybridization were kept identical, and experiments differed only in the method in which dsRNA was applied to the membranes (dot-blot or Northern blot). The Northern blot hybridization method yielded more consistent results, and it was visually and unequivocally shown to which dsRNA segment a cDNA probe bound, since the dsRNA segments were separated by PAGE prior to blotting and hybridization. In contrast, the dot-blot hybridization method gave less consistent results. Identical results for plasmid and insert probes were obtained for Northern blot hybridizations but not for dot-blot hybridizations. At least two probes could be used simultaneously in Northern blot hybridizations.

Blotting, Northern↗
PubMed1996

Northern blotting.

Northern blotting is the main method for examining the expression of genes through measurement of an mRNA. Tissue specificity and the factors which regulate expression can both be determined by Northern blotting. A cDNA radioactively labelled with 32P is the most-commonly-used hybridization probe. Convenient, non-radioactive detection protocols are, however, increasingly available. A combination of anti-sense oligonucleotides as probes, together with chemiluminescence-based detection provides a rapid and simplified approach to Northern blotting, increasing the accessibility of this important procedure for nutritional studies.

Blotting, Northern↗
PubMed1986

Human acid beta-glucosidase: Northern blot and S1 nuclease analysis of mRNA from HeLa cells and normal and Gaucher disease fibroblasts.

Human acid beta-glucosidase (beta-Glc) mRNA was evaluated by dot blot, Northern blot, and S1 nuclease analyses of extracts of HeLa cells and cultured fibroblasts from normal individuals and Gaucher disease patients. Dot blot quantitation indicated an equal concentration of specific mRNA in all sources. Northern blot analyses demonstrated the presence of three poly (A)+ mRNAs of about 5,600, 2,500, and 2,000 nucleotides in length from all cell extracts. All three mRNAs were present in normal amounts in fibroblast extracts from several subtypes and variants of Gaucher disease. The largest poly (A)+ mRNA species was thought to represent an unspliced nuclear precursor for the two smaller beta-Glc mRNAs. S1 nuclease analyses, using SP6 transcripts of beta-Glc cDNA, indicated that the 2,000 nucleotide mRNA differs from the 2,500 nucleotide form at both the 5' and 3' ends. These results are consistent with the use of an alternate 5' splice site and 3' polyadenylation signal. These results also suggest that the subtype and variants of Gaucher disease result from single base alterations that lead to the synthesis of defective beta-Glc.

Cells, Cultured↗
PubMed1988

Heterogeneous expression of glutamine synthetase mRNA in rat liver parenchyma revealed by in situ hybridization and Northern blot analysis of RNA from periportal and perivenous hepatocytes.

Using radiolabeled specific cDNA glutamine synthetase mRNA could be detected by in situ hybridization exclusively within those few perivenous hepatocytes which stained immunocytochemically for glutamine synthetase. This localization of glutamine synthetase mRNA was recently reported by Moorman et al. [(1988) J. Histochem. Cytochem. 36, 751-755]. Biotinylated cDNA was not suitable for mRNA detection because of a very high background staining under the conditions of in situ hybridization. Dot blot and Northern blot analysis of RNA isolated from periportal and perivenous subfractions of hepatocytes also demonstrated the exclusive perivenous localization of two hybridizable glutamine synthetase mRNAs of length 2.8 and 1.6 kilobases. These results indicate that the unique heterogeneity of glutamine synthetase in rat liver parenchyma is controlled at the pretranslational level.

Animals↗
PubMedDate not supplied

Prognostic significance of the combined expression of matrix metalloproteinase-9, urokinase type plasminogen activator and its receptor in breast cancer as measured by Northern blot analysis.

Using Northern blot analysis we have measured the co-expression of the matrix metalloprotease MMP-9, plasminogen activator urokinase type (uPA) and its receptor (uPAR) mRNAs in 81 biopsies of breast carcinomas with the objective of analyzing the impact of these factors on the overall survival probability of the patients (median follow-up time: 4 years). Individual mRNA levels of either uPA or uPAR showed parallel variations with MMP-9 mRNA, suggesting a coordinate transcription of these markers. When median values were used as cutoff points to discriminate between high and low factor expression, no association was found with patient outcome and MMP-9 or uPA mRNA distribution. However, increased uPAR mRNA levels were associated with poor prognosis (p = 0.01). The combination of MMP-9 and uPAR mRNA measurements has not enhanced prognostic information compared to information supplied by the receptor alone (p = 0.01). The combination of MMP-9 and high levels of uPA mRNA led to a significant association with poor outcome (p = 0.03). Multivariate analysis supported the notion that increased uPAR mRNA production in primary breast cancer may be a predictor of overall survival.

Adult↗
PubMed1999

Verification of differential gene transcription using virtual northern blotting.

We introduce here an alternative to conventional northern blotting that requires only minute amounts of RNA. This has been achieved by modification of methods currently used for the mapping of mRNA 5'-terminal ends. The terminal desoxynucleotidyl transferase-mediated G-tailing, cap finder, ligation-anchored and RNA ligase-mediated approaches followed by polymerase chain reaction protocols all produced high quality cDNAs in large amounts. These cDNAs could be separated by electrophoresis to obtain virtual northern blots that could replace conventional northern blots. All the essential information, including transcript length and the expression pattern, are preserved in these cDNAs, even if the transcripts are long or GC-rich. In addition, minute amounts of material (less than 100 cells) are sufficient to produce more than 100 virtual northern blots, making this approach extremely versatile.

Animals↗
PubMed1993

Expression of the cysteine proteinase inhibitor cystatin C gene in rat heart: use of digoxigenin-labeled probes generated by polymerase chain reaction directly for in situ and northern blot hybridizations.

Cystatins represent a widely distributed superfamily of cysteine proteinase inhibitory proteins. We investigated the expression of the cystatin C gene, belonging to the family 2 of cystatins, in the hearts of female rats. Using a highly sensitive reverse transcriptase-polymerase chain reaction (RT-PCR) we have detected cystatin C mRNA in the ventricule and atrium, as well as in liver and submandibular gland. A digoxigenin-labeled cystatin C probe, generated by PCR, hybridized to a single mRNA species of about 700 nucleotides on Northern blots. Northern blot hybridizations established that neither an acute inflammation produced by injection of turpentine nor administration of the beta-adrenergic agonist isoproterenol had an effect on the level of cystatin C mRNA in the heart. In situ hybridizations with digoxigenin-labeled probe localized the expression of the cystatin C gene to cardiac muscle fibers but not to other cardiac cellular elements. Cystatin C may be released by cardiac muscle fibers under physiological and pathological conditions and may modify inflammatory and necrobiotic processes.

Animals↗
PubMed2025

Northern Blotting: Protocols for Radioactive and Nonradioactive Detection of RNA.

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 32P-labelled probes, the second based on nonradioactive digoxigenin-labelled probes.

Blotting, Northern↗
PubMed1993

Quantitative and sensitive northern blot hybridization using PCR-generated DNA probes labeled with digoxigenin by nick translation.

Northern blot hybridization is one of the most convenient methods of detecting an mRNA. Nonradioactive Northern blotting using digoxigenin (DIG) is becoming widely applied because it is rapid and safe. Previous studies have indicated that DIG-labeled RNA probes are suitable for Northern blot hybridization. Here, the application of PCR-generated double-stranded DNA probes labeled with DIG by nick translation is described. DNA probes were synthesized by PCR, then labeled with DIG by nick translation. Northern blot hybridization was performed using the DIG-labeled DNA probes, and the signals were detected by means of a chemiluminescent reaction. A low amount of DIG-dUTP in the labeling reaction resulted in excellent Northern blots with low background. Densitometric analysis of the blots showed that the mRNA concentrations could be determined by densitometric analysis. The sensitivity of the DIG-Northern system was comparable to Northern blotting using 32P and was sufficiently sensitive to detect low-abundance mRNA.

Animals↗
PubMed1993

In situ hybridization and northern blot demonstration of HLA-G mRNA in human trophoblast populations by locus-specific oligonucleotide.

Trophoblast cells from normal first trimester pregnancies have been shown to express the nonclassical class I molecule, HLA-G, which is nonpolymorphic and has a heavy chain of 40 kD. In this study, in situ hybridization experiments were performed using a biotinylated HLA-G specific oligonucleotide on sections of normal placenta including the implantation site. HLA-G mRNA was identified in all extravillous trophoblast populations including the cytotrophoblast cell columns, interstitial trophoblast, endovascular trophoblast, and placental bed giant cells. In addition, villous cytotrophoblast and villous mesenchymal cells also contained HLA-G transcripts, but villous syncytiotrophoblast was always negative.

Blotting, Northern↗
PubMed1993

Detection of 5-hydroxytryptamine2 receptors by radioligand binding, northern blot analysis, and Ca2+ responses in rat primary astrocyte cultures.

Radioligand binding, Northern blot analysis, and changes in [Ca2+]i were used to study serotonin [5-hydroxytryptamine (5HT)] receptor subtypes in primary cultures of astrocytes from neonatal rat cerebral cortex. Radioligand binding studies revealed the presence of 5HT2, but not the 5HT1 or 5HT3 receptor subtypes. Radioligand binding was also used to show the presence of serotonin uptake sites, which had previously been shown to be present by [3H]-5HT uptake, and also alpha 1-adrenergic receptors as has previously been reported by binding studies. Northern blot analysis of cortical astrocyte mRNA demonstrated the presence of transcripts for 5HT2 receptors, but failed to identify mRNA for 5HT1a or 5HT1c receptors. Thus, results from Northern blot analysis correlated with the radioligand binding data which showed only 5HT2 receptors. Equilibrium saturation studies, using 125[I]-LSD to label 5HT2 receptors, yielded a KD of 9 nM and a Bmax of 177 fmol/mg protein. Radioligand binding studies or primary astrocyte cultures prepared from other brain regions also showed the presence of alpha 1-adrenergic, 5HT2 receptor, and 5HT-uptake sites, but no detectable 5HT1a receptors, which were the only 5HT1 receptors studied. Studies demonstrating 5HT-induced, spiperone- and ketanserin-sensitive increases in free [Ca2+]i as measured by FURA-2, showed that the 5HT2 receptors were functional in these cells. These data provide clear evidence for the existence of both 5HT2 receptors and 5HT-uptake sites in the same primary astrocyte cultures from neonatal rat cerebral cortex, with no detectable evidence of 5HT1a or 5HT1c subtypes.

Animals↗
PubMed1998

Nonradioactive Northern blot analysis of plant RNA and the application of different haptens for reprobing.

Northern blot analysis using radioactive probes is still the most common technique to determine the accumulation of transcripts in cells and tissues. The main disadvantages of this technique are the possible health hazard, inconvenience during handling, the high amount of RNA target necessary for detection, and difficulties with stripping and reprobing. In this paper, we propose an easily applicable protocol for Northern blot analysis of plant RNA with enhanced sensitivity using digoxigenin-, fluorescein-, or biotin-labeled in vitro transcripts derived from PCR products. Furthermore, we show the rehybridization of Northern blots with fluorescein as a second hapten, avoiding any stripping procedure.

Biotin↗
PubMed1991

Use of riboprobes for northern blotting analysis.

We compared cDNA and riboprobes for their use in Northern blotting analysis and found that higher hybridization signal can be obtained using riboprobes. Comparison of five nylon membranes for Northern blotting using riboprobe revealed that each nylon membrane differed in their performance. Background noise was minimal for all five membranes for two riboprobes. However, because of the higher thermal stability of RNA.RNA hybrids, the riboprobes could not be stripped out from the membrane even after five hours of treatment at 80 degrees C.

Animals↗
PubMed1995

A reliable and sensitive method for non-radioactive northern blot analysis of nerve growth factor mRNA from brain tissues.

This report describes a sensitive, rapid and reproducible protocol for non-isotopic Northern blotting analysis. Church buffer and probes labeled with digoxigenin (DIG) were used for studying the expression of nerve growth factor (NGF) in the rat brain. Using the described method, NGF cRNA probe was hybridized to blotted RNA and the results were compared to Northern blot obtained using the method recommended by the manufacturer (Boehringer Mannheim). Comparison revealed that the blot treated with Church buffer detected at least 10-fold more NGF mRNA as compared to blot hybridized with formamide buffer. In summary, we have developed an optimal hybridization protocol to perform non-radioactive Northern blot analysis using antisense RNA as a probe. This method allowed us to detect the specific low-abundant mRNA and analyze the expression of neurotrophic factors in the rat brain.

Animals↗
PubMed1998

Nonradioactive Northern blotting with biotinylated and digoxigenin-labeled RNA probes.

The application of nonradioactive RNA probes for Northern blotting offers the advantage of a rapid turn-around time for results without the loss of sensitivity for target mRNA detection. However, a problem that has impeded the widespread use of nonradioactive RNA probes for use in Northern blotting is the difficulty in stripping these probes from nylon membranes after hybridization. In this report we describe two protocols for stripping digoxigenin (Dig)-labeled RNA probes from nylon membranes. One protocol utilizes a phosphate-buffered formamide stripping solution to remove nonchemically modified (regular) RNA probes while the other method utilizes strippable probes that were produced with a chemically modified nucleotide (CTP) and removed by a specific stripping solution. This latter method was developed by Ambion Inc. and is called Strip-EZ. We also describe a protocol for the detection of two separate rat mRNAs using both biotin and digoxigenin-labeled RNA probes that does not require stripping the membrane after hybridization. Finally, we describe the use of another new labeling technology, called Chem-Link, that quickly and conveniently labels RNA for use in Northern blotting.

Animals↗