Improved non-radioactive Northern blot protocol for detecting low abundance mRNAs from mammalian tissues

A rapid and sensitive non-radioactive Northern blot protocol is described which has been optimised in several critical steps. This is based on a formaldehyde-denaturing agarose gel electrophoresis, an alkaline transfer, hybridisation with digoxigenin-DNA probes and detection with a chemiluminescent substrate. This method allows even low abundance mRNAs to be detected in total RNA samples from mammalian tissues.     

Expression and localization of the CYP2B subfamily predominantly in neurones of rat brain

Despite the very small amounts of cytochrome P450 (P450, CYP) enzymes expressed in different areas and cell populations of the brain as compared with the liver, there is significant evidence for their specific involvement in brain development, function and plasticity. Nevertheless, the current discussion about occurrence and importance of cerebral cytochrome P450s is determined by inconsistent interpretations of their function in general and with respect to single isoforms. Continuing a series of publications about brain P450 isoforms, we now present evidence for the constitutive expression of CYP2B1 and CYP2B2 mRNAs in rat brain. Immunocytochemical and non-radioactive in situ hybridization studies revealed the same expression pattern throughout the brain predominantly in neuronal populations, but to some extent in astrocytes of corpus callosum and olfactory bulb. The well known testosterone-metabolizing capacity and the presence of CYP2B isoforms shown in steroid hormone-sensitive areas and neurones (e.g. hippocampus) clarify the significance of isoforms like CYP2B1 and CYP2B2 for impairment of steroid hormone actions by P450 inducing environmental substances. We argue that cerebral P450 isoforms which are induced by xenobiotics and are able to metabolize these as well as endogenous substrates help us to understand fundamental aspects of brain's functioning.     

A New Efficient Stereoselective Method for the Synthesis of (E)-5-Aminoallyl-Pyrimidine-5′-Triphosphates Using Palladium-Catalyzed Heck Reaction

An efficient overall two-step strategy for the synthesis of (E)-5-aminoallyl-pyrimidine-5′-triphoshate, starting from commercially available pyrimidine-5′-triphosphate is described. The method involves regioselective iodination of pyrimidine-5′-triphosphate, followed by the palladium-catalyzed Heck coupling with allylamine. The catalytic reaction is highly stereoselective and compatible with many functional groups present in the reactants.     

A Comprehensive Analysis of Northern versus Liquid Hybridization Assays for mRNAs,Small RNAs,and miRNAs Using a Non-Radiolabeled Approach

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.     

Detection of citrus yellow mosaic virus by PCR and nucleic acid spot hybridisation using non-radioactive probes in commercial citrus species

Citrus yellow mosaic virus (CYMV) was detected by polymerase chain reaction (PCR) in leaf samples of sweet orange, Rangpurlime, Pumello pink and acid lime and also in twig bark, fruit rind, fruit juice except fruit rag of sweet orange cv. Sathgudi, where all the positive samples showed bright amplification of the 726 bp band except in fruit rind. The CYMV could be detected by the biotin labelled probe of CYMV up to 1:100 dilutions by TE and TNE extraction methods in sweet orange cv. Sathgudi as well as Rangpurlime.     

用非放射性原位杂交在同一张切片上同时在光学和电子显微镜下显示的豌豆rDNA的分布(简报)

原位杂交目前已成为研究各类组织或细胞内DNA或RNA定位最有效的手段,特别是在发育生物学领城,对于研究基周的表达和修饰更具有重要意义。旱期的原位杂交主要是在     

Use of non-radioactive digoxigenin-labeled DNA probes for RFLP analysis in rice

A simple procedure for the detection of rice RFLPs with non-radioactive probes is described. Rice single-copy DNA was labeled with non-radioactive digoxigenin-d UTP. When digested total DNA was hybridized with the non-radioactive labeled DNA probes, RFLPs for rice single-copy DNA could be successfully detected.     

Use of image analysis software as a tool to visualize non-radioactive signals in plantin situ analysis

In situ hybridization (ish) allows the visualization of gene expression in tissues at high microscopic resolution. Interference by plant tissue pigments generally confers higher sensitivity to radioactiveish, relative to non-radioactiveish using hapten labeled probes. The increased resolution is partially due to image acquisition methods in radioactiveish experiments. However, radioactiveish has many drawbacks including short probe life, safety concerns associated with the use of radioactive materials, and slow development of signal. In this report, we show how commercially available image analysis software can be used to extract data from non-radioactiveish images to gain a substantial increase in resolution. We provide a comparison between detecting a probe (CELLULOSE SYNTHASE) that is expected to produce a consistent, detectable signal in all growing tissues with detection of a probe (LEAFY)that is expected to produce a signal only in specific tissues. Although the scientific content of this article has been reviewed,the full-text Web publication has not been edited in detail.