Quantitative molecular hybridization with unfractionated, solubilized cells using RNA probes and polyacrylamide gel electrophoresis

Molecular hybridization with RNA probes was performed on unfractionated cells solubilized in guanidine thiocyanate solutions. Unhybridized probe was digested with ribonuclease, and protected probe fragments were resolved by polyacrylamide gel electrophoresis (PAGE). Since the same medium was used both for solubilization of the cells and as the hybridization buffer, RNA purification was not required and the analysis of large numbers of samples was facilitated. Using this method, specificity is superior to dot blot analysis because the size of hybridized fragments is determined and the signal of the probe hybridized to target RNA is separated from the background by PAGE.     

In situ hybridization using PEG-embedded tissue and riboprobes: increased cellular detail coupled with high sensitivity

We describe a procedure for preparing tissue sections by embedding in polyethylene glycol for subsequent in situ hybridization analysis using single-stranded RNA probes. Improved tissue morphology is obtained as compared to frozen sections, and the embedding procedure is milder and faster than paraffin embedding. Sections as thin as 2 microns are readily cut from PEG-embedded brain tissue. A simplified hybridization protocol (Clayton et al.: Neuron 1:249, 1988) supports the detection of even low-abundance brain mRNAs (less than or equal to 10(-4) fractional mRNA mass). By employing high stringency washes in place of ribonuclease treatment after hybridization, cell RNA is retained for cresyl violet staining, and high signal:noise ratios are achieved. Solutions to problems with section mounting and adherence to glass slides are presented. The combination of improved morphology, high signal levels, and relative simplicity should make this procedure useful in a variety of applications.     

Enhanced RNA cleavage within bulge-loops by an artificial ribonuclease

Cleavage of phosphodiester bonds by small ribonuclease mimics within different bulge-loops of RNA was investigated. Bulge-loops of different size (1–7 nt) and sequence composition were formed in a 3′ terminal fragment of influenza virus M2 RNA (96 nt) by hybridization of complementary oligodeoxynucleotides. Small bulges (up to 4 nt) were readily formed upon oligonucleotide hybridization, whereas hybridization of the RNA to the oligonucleotides designed to produce larger bulges resulted in formation of several alternative structures. A synthetic ribonuclease mimic displaying Pyr–Pu cleavage specificity cleaved CpA motifs located within bulges faster than similar motifs within the rest of the RNA. In the presence of 10 mM MgCl₂, 75% of the cleavage products resulted from the attack of this motif. Thus, selective RNA cleavage at a single target phosphodiester bond was achieved by using bulge forming oligonucleotides and a small ribonuclease A mimic.     

Isolation of RNA for dot hybridization by heparin-DNase I treatment of whole cell lysate

We have developed a new procedure for the rapid preparation of undegraded total RNA from cultured cells for specific quantitation by dot blotting analysis. Pelleted cells are resuspended in hypotonic solution containing a ribonuclease inhibitor and heparin and disrupted by freeze-thaw. Heparin is employed as an agent for nuclear lysis, dissociation of chromosomal protein, and release of mRNA from rough endoplasmic reticulum. We eliminate chromosomal DNA by digestion with DNase I and denature the RNA in the lysate with formaldehyde. After centrifugation to remove debris, the supernatant is used directly for dot blotting. All manipulations are performed in the same microfuge tube and recovery of RNA is quantitative. The procedure is especially useful for processing large numbers of samples. We illustrate its versatility by analysis of specific RNAs in Drosophila, rat, and human cell lines. In reconstruction experiments, less than 80 molecules per cell of a small RNA (beta-globin) can be detected under highly stringent hybridization conditions, using only moderately labeled double-stranded plasmid DNA probes and short film exposures.     

Linear 2' O-Methyl RNA probes for the visualization of RNA in living cells

U1snRNA, U3snRNA, 28 S ribosomal RNA, poly(A) RNA and a specific messenger RNA were visualized in living cells with microinjected fluorochrome-labeled 2' O-Methyl oligoribonucleotides (2' OMe RNA). Antisense 2' OMe RNA probes showed fast hybridization kinetics, whereas conventional oligodeoxyribonucleotide (DNA) probes did not. The nuclear distributions of the signals in living cells were similar to those found in fixed cells, indicating specific hybridization. Cytoplasmic ribosomal RNA, poly(A) RNA and mRNA could hardly be visualized, mainly due to a rapid entrapment of the injected probes in the nucleus. The performance of linear probes was compared with that of molecular beacons, which due to their structure should theoretically fluoresce only upon hybridization. No improvements were achieved however with the molecular beacons used in this study, suggesting opening of the beacons by mechanisms other than hybridization. The results show that linear 2' OMe RNA probes are well suited for RNA detection in living cells, and that these probes can be applied for dynamic studies of highly abundant nuclear RNA. Furthermore, it proved feasible to combine RNA detection with that of green fluorescent protein-labeled proteins in living cells. This was applied to show co-localization of RNA with proteins and should enable RNA-protein interaction studies.     

Linear 2′ O-Methyl RNA probes for the visualization of RNA in living cells

U1snRNA, U3snRNA, 28 S ribosomal RNA, poly(A) RNA and a specific messenger RNA were visualized in living cells with microinjected fluorochrome-labeled 2′ O-Methyl oligoribonucleotides (2′ OMe RNA). Antisense 2′ OMe RNA probes showed fast hybridization kinetics, whereas conventional oligodeoxyribonucleotide (DNA) probes did not. The nuclear distributions of the signals in living cells were similar to those found in fixed cells, indicating specific hybridization. Cytoplasmic ribosomal RNA, poly(A) RNA and mRNA could hardly be visualized, mainly due to a rapid entrapment of the injected probes in the nucleus. The performance of linear probes was compared with that of molecular beacons, which due to their structure should theoretically fluoresce only upon hybridization. No improvements were achieved however with the molecular beacons used in this study, suggesting opening of the beacons by mechanisms other than hybridization. The results show that linear 2′ OMe RNA probes are well suited for RNA detection in living cells, and that these probes can be applied for dynamic studies of highly abundant nuclear RNA. Furthermore, it proved feasible to combine RNA detection with that of green fluorescent protein-labeled proteins in living cells. This was applied to show co-localization of RNA with proteins and should enable RNA–protein interaction studies.     

Poly(A) RNA codistribution with microfilaments: evaluation by in situ hybridization and quantitative digital imaging microscopy

The distribution of poly(A) RNA has been visualized in single cells using high-resolution fluorescent in situ hybridization. Digital imaging microscopy was used to quantitate the signal in various cellular compartments. Most of the poly(A) signal remained associated with the cellular filament systems after solubilization of membranes with Triton, dissociation of ribosomes with puromycin, and digestion of non-poly(A) RNA with ribonuclease A and T1. The actin filaments were shown to be the predominant cellular structural elements associating with the poly(A) because low doses of cytochalasin released about two-thirds of the poly(A). An approach to assess the extent of colocalization of two images was devised using in situ hybridization to poly(A) in combination with probes for ribosomes, membranes, or F-actin. Digital imaging microscopy showed that most poly(A) spatially distributes most significantly with ribosomes, slightly less with F-actin, and least of all with membranes. The results suggest a mechanism for anchoring (and perhaps moving) much of the cellular mRNA utilizing the interaction between actin filaments and poly(A).     

Purification and analysis of RNA from paraffin-embedded tissues

We have developed methods for the purification and analysis of RNA from formalin-fixed and paraffin-embedded tissue. The methods allow retrospective analysis of gene expression or viral infection. RNA extraction uses prolonged solubilization of tissue with detergent and protease in the presence of high concentrations of a ribonuclease inhibitor. The purified RNA is moderately degraded but its hybridization behavior is essentially unaffected. We were able to quantify specific mRNAs by dot-blot hybridization.     

Ensuring recovery of intact RNA from rat pancreas

The isolation of intact RNA from rat pancreas is compromised by autolysis and by the presence of endogenous ribonucleases. In order to ameliorate recovery we systematically investigated available RNA extraction methods and paid particular attention to the influence of frozen storage and ribonuclease inhibition strategies on overall yield and quality of RNA. Modifications to the basic procedure of Chomczynski and Sacchi (1987) are described which allow, reproducibly, to obtain rat pancreatic RNA suitable for Northern blot hybridization, RT-PCR, and differential display analysis.     

Studies on the conformation of the 3' terminus of 18-S rRNA

We have studied the conformation of the 3' end of 18-S RNA from human, hamster and Xenopus laevis cells. The 3'-terminal oligonucleotide in a T1 ribonuclease digest of 18-S RNA from HeLa cells was identified, using a standard fingerprinting method. The sequence (G)-A-U-C-A-U-U-A, established by Eladari and Galibert for HeLa 18-S rRNA, was confirmed. An identical 3' terminus is present in hamster fibroblasts and Xenopus laevis cells. The ease of identification of this oligonucleotide has enabled us to quantify its molar yield relative to several other oligonucleotides, and hence to analyse the 3' terminus by several conformation probes. Its sensitivity to S1 nuclease, limited T1 ribonuclease digestion, bisulphite modification and carbodiimide modification was consistent with the terminal oligonucleotide being in a highly exposed conformation. The m6/2A-m6/2A-C-containing sequence of 18-S rRNA also appears to be in an exposed location on the basis of three of these probes.     

"Run-off" synthesis and application of defined single-stranded DNA hybridization probes.

A simple and efficient method for synthesizing radioactively labeled single-stranded DNA hybridization probes with Thermus aquaticus (Taq) DNA polymerase is described. This is done in a "run-off" polymerization with repeated cycles of denaturation, annealing, and extension. It leads to high yields of a single-stranded DNA of defined length (up to 5000 nt), which is labeled to a high specific activity (1.3 x 10(8) cpm/micrograms DNA). These hybridization probes are equally sensitive as nick-translated DNA probes, but strand specific. This was tested by slot blot hybridization with in vitro-transcribed target RNAs and by Northern blotting. The use of single-stranded DNA hybridization probes combines the benefits of DNA stability and single-strand RNA probes.     

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.     

Quantitative Determination and Location of Newly Synthesized Virus-Specific Ribonucleic Acid in Chicken Cells Infected with Rous Sarcoma Virus

A sensitive and quantitative nucleic acid hybridization assay for the detection of radioactively labeled avian tumor virus-specific RNA in infected chicken cells has been developed. In our experiments we made use of the fact that DNA synthesized by virions of avian myeloblastosis virus in the presence of actinomycin D (AMV DNA) is complementary to at least 35% of the sequences of 70S RNA from the Schmidt-Ruppin strain (SRV) of Rous sarcoma virus. Annealing of radioactive RNA (either SRV RNA or RNA extensively purified from SRV-infected chicken cells) with AMV DNA followed by ribonuclease digestion and Sephadex chromatography yielded products which were characterized as avian tumor virus-specific RNA-DNA hybrids by hybridization competition with unlabeled 70S AMV RNA, equilibrium density-gradient centrifugation in Cs(2)SO(4) gradients, and by analysis of their ribonucleotide composition. The amount of viral RNA synthesized during pulse labeling with (3)H-uridine could be quantitated by the addition of an internal standard consisting of (32)P-labeled SRV RNA prior to purification and hybridization. This quantitative assay was used to determine that, in SRV-infected chicken cells labeled for increasing lengths of time with (3)H-uridine, labeled viral RNA appeared first in a nuclear fraction, then in a cytoplasmic fraction, and still later in mature virions. This observation is consistent with the hypothesis that RNA tumor virus RNA is synthesized in the nucleus of infected cells.