检测烟草中烟草花叶病毒的RNA斑点杂交法

用普通烟草花叶病毒OM株3′-端约2kb的cDNA为探针,探索了用RNA斑点杂交法对烟草组织中烟草花叶病毒RNA进行检测的条件。这些条件包括用分子杂交法观察云南烟区和上海烟草上分到的烟草花叶病毒与OM株的同源性,从烟草组织中提取烟草花叶病毒的几种方法的比较,使RNA有效地固定在硝酸纤维素滤膜上的方法,烟草组织中是否有干扰RNA固定和杂交的物质,斑点杂交方法检测烟草花叶病毒的特异性、灵敏度等。     

A new sensitive radial diffusion method for microdetermination of alpha-amylase

A rapid, convenient, and efficient hybridization method for the determination of virus-specific RNAs in Py virus-infected cells is described. The method involves carrying out the hybridization of viral RNAs present in the RNA isolated from infected cells directly on nitrocellulose filters carrying denatured, immobilized Py-DNA (15 μl RNA solution/25-mm² filter). Under optimal conditions quantitative hybridization of viral RNA sequences is obtained within 24 h. The efficiency of hybridization is increased significantly when RNA fragmented by alkali under carefully controlled conditions is used.     

Restriction endonuclease mapping by crossed contact hybridization: the ribosomal RNA genes of Achlya ambisexualis.

A rapid, convenient and economical method for the hybridization of electrophoretically resolved RNA to DNA restriction fragments immobilized on nitrocellulose filters is described. DNA was digested, electrophoresed on agarose gels in a wide band and transferred to a nitrocellulose filter. The filter was then placed on the surface of a second gel containing radioactively labeled RNA electrophoresed under denaturing conditions in a similar way. The filter and gel were oriented so that the DNA and RNA bands were perpendicular to one another and the RNA was transferred from the gel through the filter under conditions which promote RNA-DNA hybridization. Following washing, the filter was autoradiographed. RNA-DNA sequence relationships could be conveniently determined from the spots produced at regions of intersection of homologous nucleic acids. The two dimensional array formed in this procedure fascilitates the rapid ordering of DNA restriction fragments. An example of its use for this purpose is presented.     

A new method of DNA and RNA transfer onto nitrocellulose filters during blot hybridization

A quick (1-2 hour) method of DNA and RNA transfer onto nitrocellulose filters for subsequent blot-hybridization was elaborated. The main features of the method proposed are, firstly, almost complete exclusion of the mechanical impact on the gel and, secondly, addition to the transfer medium (20 X SSC) of a chaotropic agent, 0.5 M NaClO4. The latter results in a slight dissolution of the gel matrix and, on the other hand, somewhat increases the binding of the nucleic acid to the nitrocellulose. The method shortens significantly the time of DNA or RNA transfer at equal, or even higher, quality of hybridization.     

Rapid quantitation of individual RNA species in a complex population.

Many investigations require quantitation of one or more individual RNA species in complex populations. Existing methods are tedious when multiple samples are to be assayed. A method is presented which allows rapid and accurate quantitation of many species of RNA simultaneously. Recombinant plasmids containing cDNA inserts are electrophoresed in agarose and blotted to nitrocellulose. After hybridization with labeled RNA and autoradiography, bands are quantitated by scanning. The results were calibrated by solution hybridization. The approach has been validated through the use of plasmids containing inserts of Drosophila cDNA and RNA of cultured cells.     

Detection of tissue-specific gene expression using an in situ hybridization method of histoblotting

A version of in situ hybridization on the histological sections that is used for screening specific mRNA in tissues in proposed. Sections of the frozen tissue samples are prepared on the cryostatic microtome, placed on nitrocellulose filters and hybridized with labelled DNA-probes under the conditions of RNA blot hybridization. The proposed method ("histoblotting") was used to study the distribution of actin and alpha-phetoprotein mRNA genes in tissues of 15 and 21-day rat embryos. The possibility of studying mRNA (by hibridizations) and protein (by immunoenzyme staining) and making histological analysis simultaneously by histoblotting is demonstrated.     

An assay for quantitative nucleic acid hybridization on membrane filters

A quantitative nucleic acid hybridization assay based on a 6-mm-diameter nitrocellulose membrane filter and only 20 microliters of hybridization mixture per determination is described. As a consequence of the small ratio of hybridization volume to membrane surface, the hybridization rates reached in this system are higher than those obtained at volume to surface ratios of conventional protocols, allowing even RNA at very low concentrations to complete hybridization. This advantage, together with the high reproducibility and hybrid stability obtained with the assay, increases the ability of the filter hybridization technique to analyze quantitatively minute amounts of RNA.     

SV40 RNA: Filter hybridization for rapid isolation and characterization of rare RNAs

Modifications of the filter hybridization method for the measurement and isolation of simian virus 40 (SV40) RNA from transformed cells are described. The modified method used small (0.02 cm²) nitrocellulose filters with > 30 μg/cm² SV40 DNA applied following formaldehyde denaturation. The small volume and high DNA densities allowed hybridization to be completed in 2 h and washing after hybridization to be completed in 6 h. The washing reduced background to 1 × 10^?5 of input radioactivity without using nucleases. The efficiency of hybridization after washing was 40% or greater. These procedures have allowed the quantification of proportions of SV40 RNA in labeled RNA from transformed lines and the characterization of SV40 RNAs by electrophoresis and cell-free translation. A 3.7-kb SV40 RNA from SV80 cells was discovered in this work.     

One-hour downward alkaline capillary transfer for blotting of DNA and RNA.

The downward alkaline capillary transfer of DNA and RNA from agarose gel to a hybridization membrane was performed using a transfer solution containing 3 M NaCl and 8 mM NaOH. Under mild alkaline conditions, DNA and RNA were completely eluted from the agarose gel and bound to a hybridization membrane within 1 h. On the basis of this new method of transfer a blotting protocol, downward alkaline blotting, was elaborated. It provides a fast and efficient alternative to commonly used Southern and Northern blotting protocols. The downward alkaline blotting of DNA and RNA can be completed in 2.5 and 1.5 h, respectively, and can be used with both plastic and nitrocellulose membranes. In addition, the downward alkaline blotting protocol allows for a hybridization efficiency of DNA and RNA higher than that of the standard blotting protocols performed at neutral pH.     

Determination of potato spindle tumor viroid and chrysanthenum stund viroid using biotinylated olideoxyribonucleotides]

A 26 base long oligodeoxyribonucleotide complementary to a common RNA sequence of potato spindle tuber viroid (PSTV) and chrysantemum stunt viroid (CSV) was synthesized. The 3'-end biotinylated one was used for the detection of PSTV and CSV RNA immobilized on nitrocellulose filters by nucleic acid hybridization. Visualization of hybridization results was performed by two ways, either by streptavidin-alkaline phosphatase conjugate or streptavidine and biotinylated alkaline phosphatase. It was possible to detect 0.65 ng of purified CSV and PSTV RNA. The suggested system of viroid diseases detection can be used by agricultural and horticultural enterprises.     

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.     

Purification of specific adenovirus 2 RNAs by preparative hybridization and selective thermal elution.

A method is described for the preparation isolation of highly purified adenovirus RNA species. Cytoplasmic RNAs from cells infected with adenovirus 2 were selected by hybridization to viral DNA fragments bound to nitrocellulose membranes. A series of washes at elevated temperatures (50-70 degrees) determined conditions at which the true hybrids were stable but non-specific RNA was removed. This temperature has been found to correlate with the base composition of the DNA fragment. After washing at this predetermined temperature, the specific RNA was eluted at 85 degrees. The purity of the eluted RNA was greater than 95% as determined by size, sequence specificity, and template activity in an in vitro protein synthesizing system. The method described should be generally useful for purification of specific RNAs.     

Detection of rare mRNA species in a complex RNA population by blot hybridization techniques: a comparative survey

The detection of very rare mRNA species in a complex RNA preparation by current RNA blotting techniques is not straightforward. To be able to determine the size of mRNA molecules representing 10(-6) to 10(-7) of the total mass of an RNA preparation, a quantitative comparison of the level of detection of denatured mRNA species electrophoretically separated on agarose gels, followed by transfer to either nitrocellulose or diazobenzyloxymethyl (DBM) paper and hybridization to specific cDNA probes was carried out. Different transfer procedures were analyzed. Optimal conditions have been found which allowed the detection of RNA bands containing as little as 5 pg of a specific sequence within a few days of autoradiography following hybridization with highly labeled [32P]cDNA probes. Using this procedure it was shown that the low amounts of alpha-fetoprotein (AFP) mRNA sequences present in adult rat liver are mature AFP mRNA molecules.     

Detection of avocado sunblotch viroid (ASBV) by dot-spot self-hybridization with a [32P]-labelled ASBV-RNA

RNA was extracted from plants infected with avocado sunblotch viroid (ASBV) and was analyzed by electrophoresis in polyacrylamide gel. The ASBV related fraction was eluted from the gel, labelled with [³²P] using polynucleotide kinase and used as a probe for hybridization with a purified ASBV-RNA preparation dot spotted on nitrocellulose paper. Positive self-hybridization indicated a high degree of internal complementarity. Dot spots of whole cell RNA and of leaf sap from ASBV infected plants were shown to hybridize with the labelled probe. This hybridization procedure proved to be 16–64 times more sensitive in diagnosing ASBV when compared with polyacrylamide gel analysis.     

Isolation of bacteriophage λ containing yeast ribosomal RNA genes: Screening by in situ RNA hybridization to plaques

We have developed an in situ hybridization technique which can be used to screen large numbers of hybrid bacteriophage for the presence of a particular inserted DNA sequence. Plaques of hybrid phage are formed on E. coli lawns on nitrocellulose filters, and their DNA is released, denatured, and fixed directly on the filters for hybridization to radioactive RNA probes. We have used this technique to isolate a number of hybrid bacteriophage λ which contain EcoRI restriction fragments of the ribosomal RNA genes from yeast, and have examined the DNA from several of these phage.     

Reliability of the RNA-DNA Filter Hybridization for the Detection of Oncornavirus-Specific DNA Sequences

Denatured DNA from leukemic myeloblasts or uninfected chicken embryos, immobilized on nitrocellulose filters, was hybridized to a vast excess of [(3)H]70S RNA from purified avian myeloblastosis virus. The viral RNA was eluted from the RNA-DNA hybrids, purified, and then rehybridized in solution to an excess of either leukemic or normal chicken embryonic DNA. This study revealed that all the slow and the fast hybridizing viral RNA sequences detectable by liquid hybridization in DNA excess had hybridized to the filter bound DNA. Both techniques also gave similar values for the number of 28S ribosomal RNA genes contained in a chicken cell genome: 210 by the liquid hybridization procedure and 218 by the filter hybridization technique. Therefore, filter hybridization can accurately detect DNA sequences present in relatively few numbers in the genome of higher organisms.     

Quantitative determination of mRNA phenotypes by the polymerase chain reaction

A method for quantitative determination of specific cellular mRNA is described. The mRNA in a dilution series of total RNA was reverse transcribed by an oligo-dT primer and the cDNA was amplified by the polymerase chain reaction (PCR) using sets of specific primers. A 32P- or biotin-labeled specific probe was hybridized to the PCR products immobilized on nitrocellulose membrane. The intensity of the hybridization signals was evaluated for quantification of the PCR products. A standard curve was produced by the known amount of the in vitro transcribed cRNA, which contained the same sequence as the mRNA. The series of standard cRNA dilutions were reverse transcribed, amplified and hybridized in the same manner. The amount of the specific RNA was deduced by fitting to the standard curve. Two tissue specimens of intestinal tumors, evaluated on the basis of hybridization signals by three different methods, were shown to contain similar amounts of beta-actin mRNA. Furthermore, a Chinese hamster ovary (CHO) cell line transfected with platelet-derived growth factor (PDGF) beta-receptor cDNA was found to contain similar amounts of beta-actin mRNA as the untransfected CHO cell line. However, the transfected CHO cell line contained over 10(11) copies of the PDGF beta-receptor mRNA per microgram of total RNA, while the untransfected one showed no detectable RNA, indicating that the latter contained less than 10(6) copies per microgram of total RNA in this assay.