Carbodiimide-mediated cross-linking of RNA to nylon membranes improves the detection of siRNA, miRNA and piRNA by northern blot

The northern blot, or RNA gel blot, is a widely used method for the discovery, validation and expression analysis of small regulatory RNA such as small interfering RNA (siRNA), microRNA (miRNA) and piwi-interacting RNA (piRNA). Although it is straightforward and quantitative, the main disadvantage of a northern blot is that it detects such RNA less sensitively than most other approaches. We found that the standard dose of UV used in northern blots was not the most efficient at immobilizing small RNA of 20–40nt on nylon membranes. However, increasing the dose of UV reduced the detection of miRNA by hybridization in northern blotting experiments. We discovered that using the soluble carbodiimide, EDC, to cross-link RNA to nylon membranes greatly improved the detection of small RNA by hybridization. Compared to standard UV cross-linking procedures, EDC cross-linking provided a 25–50-fold increase in the sensitivity of detection of siRNA from plants and miRNA or piRNA from mammalian cells. All types of hybridization probes tested benefited from the new cross-linking procedure. Cross-linking was dependent on a terminal phosphate and so, should be applicable to other related categories of small RNA.     

'Northern Cross' hybridization for rapid identification of exon-containing restriction fragments

The Northern Cross method allows direct comparison of restriction digests of cDNA and genomic clones to RNA populations by a specialized form of hybridization. This technique is based on the use of Northern and Southern blotting techniques and requires the use of two nylon membranes of differing chemical characteristics. A nylon membrane containing permanently affixed, electrophoretically fractionated RNAs is contact-hybridized at a right angle to a second, chemically different nylon membrane containing transiently bound, fractionated labeled DNA fragments. RNA and DNA bands possessing homology will hybridize where they cross, forming an autoradiographically detectable spot. This Northern Cross procedure proportionately represents the amounts of different RNAs derived from a particular sequence in a manner similar to what would have been observed in a Northern blot. This method, which can be used in the analysis of even relatively rare RNA species, permits rapid and fairly inexpensive identification of exon-containing fragments or determination of the relationship between related, multiple RNA species.     

Northern blot detection of endogenous small RNAs (～14 nt) in bacterial total RNA extracts

Here we describe a northern blot procedure that allows the detection of endogenous RNAs as small as ∼14 nt in total RNA extracts from bacteria. RNAs that small and as part of total bacterial RNA extracts usually escape detection by northern blotting. The approach combines LNA probes 5′-digoxigenin-endlabeled for non-radioactive probe detection with 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide-mediated chemical crosslinking of RNAs to nylon membranes, and necessitates the use of native PAGE either with the TBE or MOPS buffer system.     

Chemical Synthesis of LNA-mCTP and its application for MicroRNA detection

Locked nucleic acids (LNA) are being applied in hybridization studies, but current locked nucleotides cannot be transcribed into RNA probes. Here, the authors report the use of a new synthetic locked nucleotide, locMeCytidine-5'-triphosphate (LNA-mCTP), for hybridization study. This synthetic LNA-mCTP can be transcribed into a short ( approximately 30-nt) RNA probe. Dot blot hybridization on nylon membrane suggested that the short (33)P-LNA RNA probes had strong binding affinity to target oligonucleotides and its detection sensitivity was approximately approximately 1000 miRNAs in a 20- to 30-mum (diameter) dot area. On tissue sections, the differential expression pattern of mir-124 within different tissue regions revealed by short (33)P- LNA RNA probes correlated well to that analyzed by real-time RT-PCR. In addition, the specific cellular distribution of vasoactive intestinal polypeptide mRNAs in the mouse brain was the same using a 30-nt (33)P-LNA RNA probe and a 1.5-kb (33)P-RNA probe. These results suggested the high hybridization specificity of the small LNA-RNA probes to target small RNAs. Finally, the authors applied (33)P-LNA probes to detect miRNA let-7C expression in human cancer tissues. Let-7C was clearly present in lung, prostate, and colon cancers but undetectable in ovary and thyroid cancer samples. These results suggested that this miRNA detection method provides an alternative tool to study the cellular distribution of miRNAs in tissues.     

A procedure for Northern blot analysis of native RNA

We describe a modification of the Northern technique that allows the detection of RNA either native and/or containing hidden breaks. We found that the highest sensitivity of the hybridization signals was obtained after denaturation of the RNA in the gel prior to its transfer onto a nylon membrane (GeneScreen) followed by uv irradiation. The sensitivity of the method using native RNA was found to be equivalent to that obtained with denatured RNA.     

Insulin-like growth factor binding protein expression in human small cell lung cancer cell lines

Insulin-like growth factor binding proteins (IGF-BP) are secreted by several human small cell lung cancer cell lines (SCLC). In order to identify the IGF-BPs from SCLC cell lines the RNA from 10 different SCLC cell lines was analyzed by Northern blot analysis with the probes for three different IGF-BPs, IGFBP-1, IGFBP-2, and IGFBP-3. No hybridization signal could be detected with the probes encoding for IGFBP-1 and IGFBP-3. The hybridization with different IGFBP-2-specific oligodeoxynucleotide probes and with the corresponding full-length cDNA showed that all SCLC cell lines which secreted IGF-BPs express IGFBP-2.     

Loading and transfer control for Northern hybridization.

We report a simple and inexpensive method to quantitate loading and transfer of RNA and to examine RNA integrity for use with Northern hybridization. This technique involves quantitation by computer-assisted video densitometry of a negative photograph of 28S and 18S rRNA from ethidium bromide-stained RNA fixed to nylon membranes. This method eliminates the issue of variability of expression of "housekeeping" genes and the need for a second round of hybridization to quantitate control probes.     

Northern blot mapping: a procedure for mapping mRNA immobilized on nitrocellulose by probing with end-labeled DNA fragments

A simple method for mapping RNA on a Northern blot with a mixture of end-labeled DNA fragments is described. The DNA fragments are labeled either in 5' or in 3' directly after digestion by restriction enzyme(s) and used without any further purification step as probe to hybridize a Northern blot. After autoradiography, the DNA fragments hybridized to each mRNA species are recovered by heating the nitrocellulose and analyzed on denaturing polyacrylamide or agarose gels. This method indicates which DNA fragment hybridizes with which mRNA species and requires far fewer different manipulations than successive hybridization of a Northern blot with several nick-translated purified DNA fragments.     

Reduction of Background Problems in Nonradioactive Northern and Southern Blot Analyses Enables Higher Sensitivity Than P-Based Hybridizations

An improved chemiluminescence-based RNA/DNA detection procedure offering a widely applicable alternative to the conventional ³²P labeling employed in molecular biology is described. Even highly sensitive applications such as Northern blot analysis of low-copy RNAs are shown to be feasible now without radioactive labeling. Improved quality of nonradioactive detection was obtained by the use of digoxigenin-labeled nucleotides in combination with dioxetane substrates which are decomposed by the hydrolysis of alkaline phosphatase. Previously existing problems involving unacceptably high background signals in nonradioactive labeling procedures were eliminated by the application of a modified RNA/DNA transfer, hybridization, and detection protocol. The data presented here delineate a system consistently superior to radioactivity and should considerably increase the usefulness of nonradioactively labeled probes detected by chemiluminescence.