Sandwich hybridisation assay for quantitative detection of yeast RNAs in crude cell lysates

Background  

A rapid microtiter plate based sandwich hybridization assay was developed for detection and quantification of single RNA species using magnetic beads. Following solution hybridization target RNA molecules were collected by biotin-streptavidin affinity binding and detected by fluorescence signal generated by alkaline phosphatase. The 18S rRNA and SUC2 mRNA of Saccharomyces cerevisiae were used as model RNA target molecules.     

An improved method for the simultaneous demonstration of mRNA and esterase activity at the human neuromuscular junction

The aim of this study was to develop a simple means of studying the distribution of mRNA coding for post-synaptic proteins at the human neuromuscular junction. A reliable method by which to identify the junctions in tissue sections after in situ hybridization was essential. A method is described for combining the histochemical demonstration of esterase activity at the neuromuscular junction with autoradiographic localization of mRNA by in situ hybridization in the same cryostat section of skeletal muscle. The indigogenic esterase method of Strum and Hall-Craggs (1982) was modified in such a way that it is able to survive the multiple steps involved in in situ hybridization and autoradiography. The protocol is simple and reproducible and has been used successfully on sections of both rat and human skeletal muscle. To demonstrate the method, sections were reacted to reveal esterase activity and were then processed for in situ hybridization using a 35S-labelled probe specific for the -s ubunit of the acetylcholine receptor. The reaction product was retained after the lengthy in situ hybridization and autoradiographic procedures. To our knowledge, this is the first demonstration of acetylcholine receptor mRNA by in situ hybridization at human neuromuscular junctions. © Chapman & Hall     

一种适于植物幼胚mRNA整体原位杂交的方法

A mRNA whole-mount in situ hybridization method is reported here for quick, direct analysis of the spatial and temporal mRNA expression patterns in plant young embryos. A cDNA clone THE3 (tobacco heart embryo 3) was isolated by differential screening from tobacco (Nicotiana tabacum L.) heart embryo cDNA library as compared with the globular embryo cDNA library. The distribution of THE3 mRNA in tobacco heart embryos and globular embryos was investigated by a whole-mount in situ hybridization technique, showing that THE 3 is preferentially expressed in heart embryos.     

Applications of magnetic beads with covalently attached oligonucleotides in hybridization: isolation and detection of specific measles virus mRNA from a crude cell lysate

A novel, rapid, one-step isolation procedure utilizing oligonucleotides end-attached to magnetic beads (Dynabeads) has been developed. The beads and their captured target nucleic acids were separated after 2 h from the hybridization solution using an external magnet. This procedure was used to isolate measles virus hemagglutinin (H) mRNA from infected cells dissolved in 5 M guanidine thiocyanate. The yield of the specific, undegraded H-mRNA was found to be near the estimated total amount of H-mRNA present in the cells. The magnetic beads were also used for detection of measles virus H-mRNA in a crude cell lysate by sandwich hybridization. The experimental detection limit was 630 amol H-mRNA.     

Bacterial mRNA purification by magnetic capture-hybridization method

A magnetic capture-hybridization method was developed for purification of bacterial messenger RNA (mRNA) from total RNA by removing 5S, 16S and 23S ribosomal RNAs (rRNA). The quality of mRNA was evaluated by A(260 nm) / A(280 nm) value, denatured gel electrophoresis and RT-PCR. The results showed that highly purified and intact mRNA was obtained by this method. The magnetic capture-hybridization is a rapid and simple method for bacterial mRNA purification and has promising potential for improving studies using bacterial mRNA.     

Prolonged hybridization with a cRNA probe improves the signal to noise ratio for in-tube in situ hybridization for quantification of mRNA after fluorescence-activated cell sorting

We developed an in-tube in situ hybridization method for mRNA quantification after fluorescence-activated cell sorting (FACS-mQ). A specific RNA in a particular cell type is stained with a cRNA probe and a fluorescent dye, which allows the stained cells to be selected by FACS without excessive RNA degradation. Our previous protocol required 4 h for hybridization with a cRNA probe, which might not produce enough fluorescence signal for sorting genes with low expressions. We determined the effect of prolonged hybridization for in-tube in situ hybridization on quantitative measurement of intracellular RNAs. During the hybridization step, the quantity of ACTB mRNA decreased gradually until 4 h, but remained constant from 4 to 16 h below 63.6° C. For flow cytometry, cells hybridization with cRNA probes for TG mRNA at 60° C for 16 h showed both increased signal and decreased background fluorescence compared to those hybridized for 4 h. These results indicate that when performing in-tube in situ hybridization, hybridization temperature can be raised to 63.6° C and the hybridization step can be extended up to 16 h without excessive intracellular RNA degradation.     

Double and triple in situ hybridization for coexpression studies: combined fluorescent and chromogenic detection of neuropeptide Y (NPY) and serotonin receptor subtype mRNAs expressed at different abundance levels

Multiple fluorescence in situ hybridization is the method of choice for studies aimed at determining simultaneous production of signal transduction molecules and neuromodulators in neurons. In our analyses of the monoamine receptor mRNA expression of peptidergic neurons in the rat telencephalon, double tyramide-signal-amplified fluorescence in situ hybridization delivered satisfactory results for coexpression analysis of neuropeptide Y (NPY) and serotonin receptor 2C (5-HT2C) mRNA, a receptor subtype expressed at high-to-moderate abundance in the regions analyzed. However, expression of 5-HT1A mRNA, which is expressed at comparatively low abundance in many telencephalic areas, could not be unequivocally identified in NPY mRNA-reactive neurons due to high background and poor signal-to-noise ratio in fluorescent receptor mRNA detections. Parallel chromogenic in situ hybridization provided clear labeling for 5-HT1A mRNA and additionally offered the possibility to monitor the chromogen deposition at regular time intervals to determine the optimal signal-to-noise ratio. We first developed a double labeling protocol combining fluorescence and chromogenic in situ hybridization and subsequently expanded this variation to combine double fluorescence and chromogenic in situ hybridization for triple labelings. With this method, we documented expression of 5-HT2C and/or 5-HT1A in subpopulations of telencephalic NPY-producing neurons. The method developed in the present study appears suitable for conventional light and fluorescence microscopy, combines advantages of fluorescence and chromogenic in situ hybridization protocols and thus provides a reliable non-radioactive alternative to previously published multiple labeling methods for coexpression analyses in which one mRNA species requires highly sensitive detection.     

In situ hybridization in suspension and flow cytometry as a tool for the study of gene expression.

We describe a method to detect mRNA expression using in situ hybridization in suspension and flow cytometry. Our model was glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene expression in the leukemic cell line K562. A GAPDH cDNA probe was labeled with digoxigenin-11-dUTP and detected using an FITC-labeled anti-digoxigenin antiserum. We obtained good resolution in specific signals against background (GAPDH signal/control plasmid signal ratio +/- SE 3.5 +/- 0.9). The technique was optimized taking into account several hybridization variables, like fixation, hybridization time, effect of blocking agents, and stringency wash variations. This method also allowed us to quantitate the GAPDH RNA copy number/cell using a fluorescence standard; we obtained a figure of about 1200 copies/cell, which is in good agreement with the dot blot hybridization assay result. Flow cytometric analysis of in situ hybridization represents an original method to study gene expression. This technique has the potential to develop into a multiparametric tool for cell biology studies, examining specific mRNA production together with DNA content or membrane molecules expression, and offering the possibility to purify by sorting a cell population expressing a specific mRNA.     

Subtractive hybridization of cDNA from small amounts of plant tissue

A subtractive hybridization method is described that allows the generation of a subtractive gene library from small amounts of plant or other eukaryotic tissues. The method uses paramagnetic oligo-dT beads to capture poly-adenylated mRNA and to synthesize the complementary cDNA on a solid support. The use of magnetic beads facilitates the change of reaction buffers and the removal of primers and minimizes yield losses. Subtracted material obtained from this method can either be cloned directly or used to screen a specific library.     

A quality-controlled microarray method for gene expression profiling

Gene expression profiling on microarrays is widely used to measure the expression of large numbers of genes in a single experiment. Because of the high cost of this method, feasible numbers of replicates are limited, thus impairing the power of statistical analysis. As a step toward reducing technically induced variation, we developed a procedure of sample preparation and analysis that minimizes the number of sample manipulation steps, introduces quality control before array hybridization, and allows recovery of the prepared mRNA for independent validation of results. Sample preparation is based on mRNA separation using oligo(dT) magnetic beads, which are subsequently used for first-strand cDNA synthesis on the beads. cDNA covalently bound to the magnetic beads is used as template for second-strand cDNA synthesis, leaving the intact mRNA in solution for further analysis. The quality of the synthesized cDNA can be assessed by quantitative polymerase chain reaction using 3'- and 5'-specific primer pairs for housekeeping genes such as glyceraldehyde-3-phosphate dehydrogenase. Second-strand cDNA is chemically labeled with fluorescent dyes to avoid dye bias in enzymatic labeling reactions. After hybridization of two differently labeled samples to microarray slides, arrays are scanned and images analyzed automatically with high reproducibility. Quantile-normalized data from five biological replica display a coefficient of variation 45% for 90% of profiled genes, allowing detection of twofold changes with false positive and false negative rates of 10% each. We demonstrate successful application of the procedure for expression profiling in plant leaf tissue. However, the method could be easily adapted for samples from animal including human or from microbial origin.     

Quantitation of specific mRNA by RNA-RNA hybridization kinetics with single-stranded riboprobes

A quantitative procedure by a solution hybridization involving RNA-RNA hybridization kinetics was developed for measurement of specific mRNA accumulated in particular tissues and cells. For quantitating mouse beta-tubulin mRNA two types of riboprobes were prepared: one was a truncated RNA covering only the coding portion of beta-tubulin cDNA and the other was a non-truncated RNA covering the vector portion as well as the coding portion. These antisense RNAs were hybridized with mouse brain total cellular RNA, yielding heat-stable hybrids. Both the truncated and non-truncated antisense RNA probes showed similar hybridization kinetics. Hybridization of the sense RNA, consisting of the beta-tubulin coding portion, with the antisense RNA probe gave standards for determining the proportion of beta-tubulin mRNA in total brain RNA. By this method, the amounts of beta-tubulin mRNA included in the brains of 10- and 50-day-old mice were quantitated to be 0.0056 and 0.0011% of total RNA, respectively.     

A novel automated assay with dual-color hybridization for single-nucleotide polymorphisms genotyping on gold magnetic nanoparticle array

A high-throughput and cost-effective single-nucleotide polymorphism (SNP) genotyping method based on a gold magnetic nanoparticle (GMNP) array with dual-color hybridization has been designed. Biotinylated single-strand polymerase chain reaction (PCR) products containing the SNP locus were captured by the GMNPs that were coated with streptavidin. The GMNP array was fabricated by immobilizing single-stranded DNA (ssDNA)-GMNP complexes onto a glass slide using a magnetic field, and SNPs were identified with dual-color fluorescence hybridization. Three different SNP loci from 24 samples were genotyped successfully using this platform. This procedure allows the user to directly analyze the bead fluorescence to determine the SNP genotype, and it eliminates the need for background subtraction for signal determination. This method also bypasses tedious PCR purification and concentration procedures, and it facilitates large-scale SNP studies by using a method that is highly sensitive, simple, labor-saving, and potentially automatable.     

Molecular cloning of cDNAs for the nerve-cell specific phosphoprotein, synapsin I.

To provide access to synapsin I-specific DNA sequences, we have constructed cDNA clones complementary to synapsin I mRNA isolated from rat brain. Synapsin I mRNA was specifically enriched by immunoadsorption of polysomes prepared from the brains of 10-14 day old rats. Employing this enriched mRNA, a cDNA library was constructed in pBR322 and screened by differential colony hybridization with single-stranded cDNA probes made from synapsin I mRNA and total polysomal poly(A)+ RNA. This screening procedure proved to be highly selective. Five independent recombinant plasmids which exhibited distinctly stronger hybridization with the synapsin I probe were characterized further by restriction mapping. All of the cDNA inserts gave restriction enzyme digestion patterns which could be aligned. In addition, some of the cDNA inserts were shown to contain poly(dA) sequences. Final identification of synapsin I cDNA clones relied on the ability of the cDNA inserts to hybridize specifically to synapsin I mRNA. Several plasmids were tested by positive hybridization selection. They specifically selected synapsin I mRNA which was identified by in vitro translation and immunoprecipitation of the translation products. The established cDNA clones were used for a blot-hybridization analysis of synapsin I mRNA. A fragment (1600 bases) from the longest cDNA clone hybridized with two discrete RNA species 5800 and 4500 bases long, in polyadenylated RNA from rat brain and PC12 cells. No hybridization was detected to RNA from rat liver, skeletal muscle or cardiac muscle.     

mRNA synthesis rates in vivo for androgen-inducible sequences in mouse kidney.

A method was developed for measuring in vivo rates of mRNA synthesis in mice by pulse-labeling with the RNA precursor [3H]orotate and then using hybridization to recover specific mRNAs. The efficiency of recovery is determined with synthetic RNAs as internal hybridization standards. The method is particularly applicable to the kidney since this organ shows a strong preferential uptake of the label. Rates of synthesis, expressed as a fraction of total RNA synthesis, were measured for the androgen-inducible mRNAs coding for beta-glucuronidase (GUS), ornithine decarboxylase (ODC), the protein coded by the RP-2 gene, and the so-called kidney androgen-regulated protein (KAP). Control mRNAs coded for beta-actin, phosphoenolpyruvate carboxykinase, and major urinary protein. Testosterone markedly increased the synthesis of the androgen-inducible mRNAs, but not the control mRNAs. Induction was not seen in mutant mice lacking functional androgen receptor protein. For GUS, ODC, and RP-2 mRNAs, the fold induction of synthesis was less than the fold induction of concentration, suggesting that mRNA stabilization also plays a part in the response to androgen. For GUS, ODC, and RP-2 mRNAs, but not KAP mRNA, induction of synthesis was rapidly reversed after testosterone removal. KAP mRNA was also exceptional in that its concentration was disproportionately high compared with its rate of synthesis, implying that it is a particularly stable mRNA.     

A general method for detection and characterization of an mRNA using an oligonucleotide probe

A general method for the detection and characterization of an mRNA using an oligodeoxynucleotide probe is described. The results presented indicate that a G-dT or a dG-U base pair within a short DNA-RNA hybrid does not significantly reduce the stability of the hybrid. On this basis, we propose that 11 amino acids, including Trp and Met, can be used in selecting a peptide sequence suitable for use in designing an oligodeoxynucleotide probe complementary to a given mRNA. To test this hypothesis, we have synthesized an oligodeoxynucleotide (oligo II) complementary to the region of gastrin mRNA coding for Trp-Met-Asp-Phe and have compared its effectiveness as a hybridization probe and as a primer for the synthesis of gastrin-specific cDNA with another oligonucleotide (oligo I) complementary to the region of gastrin mRNA coding for Trp-Met-Glu-Glu. Unlike oligo I, oligo II functions as a primer in specific cDNA synthesis only when the mRNA is denatured prior to initiation of synthesis. Similarly, oligo II can be used as a specific hybridization probe for gastrin mRNA only when the RNA is denatured and partially cleaved with NaOH before hybridization. A simple procedure for denaturing gastrin mRNA to ensure efficient interaction with oligodeoxynucleotide probes is described. This procedure should be useful in studies with other oligonucleotides and mRNAs as well.