A method for obtaining high quality RNA from paraffin sections of plant tissues by laser microdissection

Laser microdissection (LM) combined with microarray analysis or next-generation sequencing of cDNA is a powerful tool for understanding molecular events in individual cell types of plants as well as animals. Obtaining high quality RNA is essential for this approach. For plant tissues, paraffin-embedded sections better preserve cell structure than do frozen sections. However, the conventional method for preparing paraffin sections is a lengthy process involving embedding the tissue and floating and drying the sections, during which time RNA degradation occurs. Here, we describe a method for preparing serial sections that greatly reduces RNA degradation: we reduced (1) the embedding time from 4–6 days to about 5 h by using a recently developed microwave method; (2) the time of floating sections from ~10 min to less than 5 min, (3) the drying time from ~12 to 1 h; and (4) the drying temperature from 42 to 4°C. With this method, we were able to isolate higher integrity RNA from many kinds of plant tissues than is typically obtained by the conventional paraffin preparation method. The improvement in RNA quality and yield removes a major obstacle to the widespread use of LM with high-throughput technologies for plants.     

Histochemical applications of two phenanthridinium compounds

The fluorescent compounds ethidium monoazide and ethidium bromide were found to react intensely with nucleic acids of fixed, paraffin embedded tissues of rat and mouse. For routine staining, 10(-5) M solutions of ethidium bromide and its monoazide analogue were virtually identical in their reactions. Fresh frozen sections of the tissues reacted in the same manner as fixed, paraffin embedded samples. Fluorescence of DNA and RNA in rat pancreas could be selectively abolished by taking advantage of the greater sensitivity of RNA to acid hydrolysis. Hydrolysis in aqueous solutions (1 N HCl at 55-60 C) abolished RNA fluorescence in 5 min, whereas 20 min or longer were required to destroy DNA fluorescence. DNA fluorescence was selectively abolished by 3 hr in 0.1 N HCl in anhydrous methanol while the RNA remained unaffected. Rat pancreas stained with the 10(-5) M ethidium compounds below pH 5.0 showed reduced RNA fluorescence, but the DNA continued to fluoresce brightly at pH 0.6. Reducing the pH of the staining solution to pH 1.0, therefore, was an additional method of selectively abolishing RNA fluorescence. Ethidium solutions in 5.0 M NaCl at pH 5.0 had little effect on DNA or RNA fluorescence. This new method of examining nucleic acids in fixed tissue samples opens new approaches to the histochemistry of these substances. The method also offers new possibilities for the study of mutagenic drug-DNA interactions.     

Histochemical Applications of Two Phenanthridinium Compounds

The fluorescent compounds ethidium monoazide and ethidium bromide were found to react intensely with nucleic acids of fixed, paraffin embedded tissues of rat and mouse. For routine staining, 10^-5 M solutions of ethidium bromide and its monoazide analogue were virtually identical in their reactions. Fresh frozen sections of the tissues reacted in the same manner as fixed, paraffin embedded samples. Fluorescence of DNA and RNA in rat pancreas could be selectively abolished by taking advantage of the greater sensitivity of RNA to acid hydrolysis. Hydrolysis in aqueous solutions (1 N HCl at 55-60 C) abolished RNA fluorescence in 5 min, whereas 20 min or longer were required to destroy DNA fluorescence. DNA fluorescence was selectively abolished by 3 hr in 0.1 N HCl in anhydrous methanol while the RNA remained unaffected. Rat pancreas stained with the 10^-5 M ethidium compounds below pH 5.0 showed reduced RNA fluorescence, but the DNA continued to fluoresce brightly at pH 0.6. Reducing the pH of the staining solution to pH 1.0, therefore, was an additional method of selectively abolishing RNA fluorescence. Ethidium solutions in 5.0 M NaCl at pH 5.0 had little effect on DNA or RNA fluorescence. This new method of examining nucleic acids in fixed tissue samples opens new approaches to the histochemistry of these substances. The method also offers new possibilities for the study of mutagenic drug-DNA interactions.     

Differentiation of DNA and RNA on filter paper discs

Differentiation of radioactive DNA and RNA deposited on filter paper discs can be accomplished by a relatively simple procedure. RNA can be efficiently removed by incubating the dises, impaled on pins, with 0.2 ml of 0.5 n NaOH for 90 min at 37°C. DNA can be removed after NaOH hydrolysis by treating the discs with 5% TCA for 30 min at 90°C. A correction is necessary to determine the actual amounts of DNA and RNA in order to account for the loss of DNA (13.8%) during the NaOH hydrolysis procedure.     

Red-blue staining of hydrolysed nucleic acids in paraffin sections

A previous treatment with 10% HC1 in tetrahydrofuran for 2-3 min at 37° C hydrolyses DNA while substantially preserving RNA in formol-fixed paraffin sections. If this treatment is followed by dyeing with basic fuchsin-thiazine or oxazine mixtures, the basic fuchsin stains DNA, the blue dye cytoplasmic RNA, though nucleolar RNA is not well preserved. A specimen sequence is to treat the hydrolysed section with a mixture of 1% aqueous trimethylthionin (Chroma), 15 ml; 0.1% basic fuchsin (G. T. Gurr), 4 ml; and glacial acetic acid, 1 ml. Stain for 15-30 min, dehydrate in acetone, then pass sections through xylene to polystyrene. The specificity of this stain for cytoplasmic RNA is sharper than that of methyl green-pyronin; hence the technic given can be a useful addition to the standard Unna-Pappenheim procedure.     

Cooperation of metal-ion fixation and target-site activation for efficient site-selective RNA scission

Iminodiacetate–DNA conjugates and acridine–DNA conjugates were synthesized and combined for site-selective RNA hydrolysis by Lu(III). When these conjugates form a ternary complex with complementary RNA, the Lu(III)–iminodiacetate complex is placed near the target phosphodiester linkage of RNA which is in front of the acridine and is activated by noncovalent interactions. The site-selective hydrolysis by these combinations is several times as fast as that achieved by combining unmodified DNA (without iminodiacetate) and the acridine–DNA conjugate.Electronic Supplementary Material Supplementary material is available for this article at .     

Quantitative microdetermination of DNA by two-dimensional electron capture gas chromatography of the thymine constituent

A highly sensitive and specific two-dimensional electron-capture gas chromatographic method has been developed for determining small amounts of DNA by measuring its thymine content. The method can also be used to measure RNA based on uracil content. The nucleic acids were hydrolyzed and released constituents were separated and detected as their chloromethyldimethylsilyl ethers. The minimal amount detected was 5 pg of each base. Standard curves were linear from 5 to 200 pg. This method allowed quantitative determination of 2 ng of DNA (routinely detectable quantity) after hydrolysis of biological material in formic acid, even in the presence of large amounts of RNA and/or protein. For example, this method has been shown to be successful in determination of the DNA contents of manually isolated nucleic such as from amphibian oocytes. Besides being accurate, the procedure was rapid: after maximal hydrolysis (usually about 45 min) the derivatization and gas chromatographic analysis was completed in another 15 min. The procedure described represents a direct biochemical alternative to cytophotometric estimation of nuclear contents and has the advantage of providing values for absolute DNA content per nucleus.     

Immunohistochemical detection of bromodeoxyuridine in formalin-fixed tissues

Summary Bromodeoxyuridine (BrdUrd) immunohisto-chemistry has recently been introduced for the visualization of DNA-synthesizing nuclei. In order to detect the BrdUrd incorporated into nuclear DNA in formalin-fixed, paraffin-embedded tissues, we tested several different pretreatment procedures including digestion with proteinase and hydrolysis with HCl, prior to immunoperoxidase staining. In order to determine the optimal conditions for detecting nuclear BrdUrd, mice were given BrdUrd and ³H-thymidine simultaneously, and the autoradiographic and immunohistochemical results obtained in BrdUrd-stained sections were compared. It was found that digestion with 0:05% proteinase at 37°C for 20 min and hydrolysis with 1 N HCl at 37°C for 20 min was sufficient to detect BrdUrd immunore-activity in ³H-thymidine-labelled nuclei, the results being virtually unaffected by the orders in which the two pretreatments were performed. Our method extends the range of application for BrdUrd, immunohistochemistry in cell-kinetic studies.     

Procedure for the base composition of RNA especially suited for samples containing relatively large amounts of DNA and protein

An inexpensive procedure suitable for determining the base composition of RNA in a sample containing large amounts of DNA and protein is presented. The method involves cold acid precipitation, mild alkaline hydrolysis, acid hydrolysis, ion-exchange separation, and spectral analysis. At least 70 μg RNA is required, which for accuracy within 7% should be no less than 4% dry weight in a chromatin sample or 2% dry weight in a ribonucleoprotein sample. Purine-pyrimidine ratios may be obtained on samples with relatively even less RNA. The precision is approximately ±2% for the values of the individual bases, and the over-all RNA recovery is 90%. Despite the multiple steps involved, three samples may be carried through the entire procedure in one day.     

Staining of Small Lymphoid Nucleoli in Paraffin Sections by Aniline-Azure B

To see small lymphoid nucleoli clearly in 1-2 μ paraffin sections, the staining of contiguous chromatin masses in the nucleus was suppressed by a hydrolysis-aniline blocking sequence, which produces aldehyde from DNA, and attaches aniline to that aldehyde to make a diphenamine base, thus reducing the acidity of the chromatin and its affinity for basic dyes. Nucleolar RNA remains fully stainable by azure B, because the hydrolysis used does not produce aldehyde groups in it, to allow aniline attachment. Technique: Hydrolyse the 10% formol-saline fixed, deparaffinised 1-2 μ section for 4.5-5.0 min in 10% (v/v) HCl in tetra-hydrofuran at 39-40 C, rinse in water, and treat at room temperature in 10% (v/v) aniline in acetic acid for 10 min. Stain 2-4 hr with freshly prepared 0.1% azure B in a 1:10 dilution of tris buffer at pH 7.0. Rinse, blot off excess water, pass through acetone and xylene to a polystyrene mounting. DNA stains pale green to colourless; nucleolar and cytoplasmic RNA, blue.     

Improved two-stage protein expression and purification via autoinduction of both autolysis and auto DNA/RNA hydrolysis conferred by phage lysozyme and DNA/RNA endonuclease

We report improved release of recombinant proteins in Escherichia coli, which relies on combined cellular autolysis and DNA/RNA autohydrolysis, conferred by the tightly controlled autoinduction of both phage lysozyme and the nonspecific DNA/RNA endonuclease from Serratia marcescens. Autoinduction occurs in a two-stage process wherein heterologous protein expression and autolysis enzymes are induced upon entry into stationary phase by phosphate depletion. Cytoplasmic lysozyme and periplasmic endonuclease are kept from inducing lysis until membrane integrity is disrupted. After cell harvest, the addition of detergent (0.1% Triton X-100) and a single 30 min freeze-thaw cycle results in >90% release of protein, green fluorescent protein. This cellular lysis is accompanied by complete oligonucleotide hydrolysis. The approach has been validated for shake flask cultures, high-throughput cultivation in microtiter plates, and larger scale stirred-tank bioreactors. This tightly controlled system enables robust growth and resistance to lysis in routine media when cells are propagated and autolysis/hydrolysis genes are only induced upon phosphate depletion.     

A Method for the Selective Removal of Deoxyribonucleic Acid from Tissue Sections

The deoxyrihonucleic acid (DNA) of chromatin undergoar depurinization on mild acid hydrolysis with a picric acid-formaldehyde mixture (Bouin's fluid). The apurinic acid thus formed is degraded by condensation with aniline and is lost from tissue sections, but ribonucleic acid (RNA) in nucleoli and cytoplasm is well preserved. Technique: Fi in Carnoy's fluid (ethanol:acetic acid 3:1 or ethanol:chloroform:acetic acid 6:3:1) or in aldehydes (10% formalin or 2.5% glutaraldehyde bsered to pH 7.0). Hydrolyse deparaEnii sections 12-24 hr at 27-50 C in Bouin's fluid, wash in distilled water, immerse in 25% (v/v) acetic acid, treat 1 hr at 27-30 C with 10% (v/v) dine in 25% acetic acid, wash in 25% acetic acid and then in water. Stain 10-40 min with 0₃% toluidine blue in 0.05 M potassium biphthalate bder (pH 4.0); rinse in distilled water, pass to 10% (w/v) ammonium molybdate for 1 min, rinse again in water and pass through tert-butanol and xylene to a synthetic resin. Chromatin and chromosomes are pale green; RNA in nucleoli and cytoplasm deep purple.     

In vivo bromodeoxyuridine incorporation in human gastric cancer: A study on formalin-fixed and paraffin-embedded sections

Summary Bromodeoxyuridine (BUDR) is a non-radioactive thymidine analogue which is incorporated into the DNA of proliferating cells. This allows evaluation of the size of the S-phase as the BUDR labelling index (BUDR-LI) not onlyin vitro but alsoin vivo, since BUDR is not toxic at the doses needed to label cells. To ascertain whetherin vivo BUDR incorporation can be detected on routine histological material we tested several different procedures prior to immunoperoxidase staining, on formalin-fixed, paraffin-embedded sections from five patients with gastric cancer, who received BUDR (250 mg m^–2, intravenous) 4 h before surgery. To determine the optimal conditions for detecting BUDR in formalin-fixed tissues, immunohistochemical testing for BUDR was performed simultaneously on duplicate sections fixed with 70% ethanol. It was found that hydrolysis with 3N HCl at 37° C for 30 min and digestion with 0.5% in at 37° C for 30 min were sufficient to detect BUDR immunoreactivity in formalin-fixed sections.The method presented extends the range of applications of thein vivo BUDR technique for cell kinetics studies in human neoplasms because it can be used on routinely fixed archival material, with the advantage of correlating the kinetic data with histopathological characters.     

A specific ultrastructural method to reveal DNA: the NAMA-Ur.

We have developed a new, simple, and reproducible cytochemical method to specifically stain DNA at the electron microscopic level: the NAMA-Ur. It is based on the extraction of RNA and phosphate groups from phosphoproteins by a weak alkali hydrolysis (NA) which does not affect DNA, followed by blockage of the amino and carboxyl groups by methylation and acetylation (MA). Finally, sections are stained by uranyl (Ur), which can bind only to DNA. The efficiency of the pre-treatment (NA and MA) was demonstrated by X-ray microanalysis at the transmission electron microscopic level. The NAMA-Ur method has been successfully performed en bloc and on Lowicryl sections in mammalian and plant cells. A specific contrast is observed in the DNA-containing structures after this method, whose sensitivity allows visualization of electron-dense chromatin fibers of 10-12 nm composed of 3-nm DNA fibrils. This staining method has been combined with anti-DNA antibodies, providing complementary information to detect DNA in situ. We propose the NAMA-Ur as an easy method to investigate the chromatin organization in situ at the ultrastructural level.     

An adaptation of the filter paper disk technique for the differential estimation of DNA and RNA of rat liver

A method is described for the differential estimation of DNA and RNA from rat liver on filter paper disks. To differentiate between DNA and RNA, samples are subjected to alkaline hydrolysis in microtitration plates prior to absorption on the disks. This procedure eliminates the loss of DNA which is observed if the disks are subjected to RNA hydrolysis after the sample has been absorbed on disks. The procedure is directly comparable to standard methods for the measurement of nucleic acids.     

Immunocytochemical detection of sulphonylated DNA in tissue sections. An alternative to Feulgen staining of DNA

We report here on a new sensitive and highly specific DNA staining technique which we have called sulpho-DNA staining. DNA staining is based on a sulphonylation reaction of 2'-deoxycytidine or cytidine that takes place in the 6th position of cytosine with ensuing immunodetection of the sulphonylated DNA. The specificity of DNA staining is introduced by the use of an antibody recognizing only modified DNA but not modified RNA, by recourse to an additional acid hydrolysis step which destroys RNA but not DNA. We describe here the optimal conditions for the sulphonylation of DNA using O-methylhydroxylamine and metabisulphite as reactants. The new DNA stain labels all nuclei in either normal human tissue or in tumor cells. For nuclear DNA the staining signal is higher for the sulpho-DNA staining than for the Feulgen staining for nuclear DNA. This new DNA staining technique is suitable for use on tissue sections as well as on cytosmears.     

Immunocytochemical detection of sulphonylated DNA in tissue sections

Summary We report here on a new sensitive and highly specific DNA staining technique which we have called sulpho-DNA staining. DNA staining is based on a sulphonylation reaction of 2-deoxycytidine or cytidine that takes place in the 6th position of cytosine with ensuing immunodetection of the sulphonylated DNA. The specificity of DNA staining is introduced by the use of an antibody recognizing only modified DNA but not modified RNA, by recourse to an additional acid hydrolysis step which destroys RNA but not DNA. We describe here the optimal conditions for the sulphonylation of DNA using O-methylhydroxylamine and metabisulphite as reactants. The new DNA stain labels all nuclei in either normal human tissue or in tumor cells. For nuclear DNA the staining signal is higher for the sulpho-DNA staining than for the Feulgen staining for nuclear DNA. This new DNA staining technique is suitable for use on tissue sections as well as on cytosmears.     

Micro-RNA quantification using DNA polymerase and pyrophosphate quantification

A rapid quantification method for micro-RNA based on DNA polymerase activity and pyrophosphate quantification has been developed. The tested micro-RNA serves as the primer, unlike the DNA primer in all DNA sequencing methods, and the DNA probe serves as the template for DNA replication. After the DNA synthesis, the pyrophosphate detection and quantification indicate the existence and quantity of the tested miRNA. Five femtomoles of the synthetic RNA could be detected. In 20–100 μg RNA samples purified from SiHa cells, the measurement was done using the proposed assay in which hsa-miR-16 and hsa-miR-21 are 0.34 fmol/μg RNA and 0.71 fmol/μg RNA, respectively. This simple and inexpensive assay takes less than 5 min after total RNA purification and preparation. The quantification is not affected by the pre-miRNA which cannot serve as the primer for the DNA synthesis in this assay. This assay is general for the detection of the target RNA or DNA with a known matched DNA template probe, which could be widely used for detection of small RNA, messenger RNA, RNA viruses, and DNA. Therefore, the method could be widely used in RNA and DNA assays.     

Cleavage of RNA in hybrid duplexes by theE. coli ribonuclease H: II. Substrate properties of oligonucleotides containing nonnucleotide linkers

The 20-mer bridged oligodeoxynucleotides containing short oligomers joined by the hexamethylenediol and hexaethylene glycol linkers were shown to form complementary DNA/DNA and RNA/DNA complexes whose thermostability depends on the length and number of the nonnucleotide linkers. Hybrid complexes of the bridged oligonucleotides proved to be substrates for theE. coli ribonuclease H. The presence of one-three nonnucleotide linkers in a 20-mer decreased the hydrolysis efficacy only 1.2–1.4-fold. It is the composition of the RNA cleavage products that was influenced the most significantly by the nonnucleotide linkers. RNase H simultaneously hydrolyzed the RNA 3′-ends of each hybrid duplex involving a bridged oligonucleotide. The presence of an inverted 3′-3′-phosphodiester bond at the 3′-end of the oligodeoxyribonucleotide only slightly affected the RNase H activity. For the previous report, see [1].