Manual in situ hybridization assay

A method for assaying target biopolymers in a celluar specimen, said method comprising the steps of:

• contacting a cellular specimen with a fixation medium comprising at least one agent that is a precipitating agent or a cross linking agent to yield a fixed spedmen having therein cells with substantially intact membranes;
• incubating said fixed Specimen with a hybridization solution comprising a denaturing agent, a hybrid stabilizing agent, a buffering agent, a membrane pore-forming agent and at least two probes each specific for different target biopolymers to be detected with each such probe having a detectable label, said incubating being under hybridizing conditions for not less than five minutes so that cellular polynucleotides of said cellular specimen have shifted to about 70% single-strandedness, wherein each detectable label is selected to be distinguishable from each other detectable label;
• and
• detectingduplexes formed between each said probe and each said different target biopolymer in said cells with substantially intact membranes by means of said detectable labels;
• wherein at least two target biopolymers are simultaneously assayed in a cell of the cellular specimen, and
• wherein the time for performing said steps of contacting, incubating, and detecting does not exceed about four hours;
• whereby said target biopolymers are detected in cells of said cellular specimen;
• wherein said shift under hybridizing conditions to about 70% single-stranded cellular polynucleotides while maintaing cellular spatial relationships is deteminable by a process comprising the following four steps:
• (a) staining cells with a stain able to stain polynucleotides within said cells to yield a different emission spectrum upon ultraviolet excitation of stained double-stranded and stained single-stranded polynucleotides;
• (b) determining the total amount of emitted fluorescence of a cell sample not treated under hybridizing conditions;
• (c) determining the total amount of emitted fluorescence of cell samples treated by various fixation and hybridization procedures and times;
• (d) assessing when the nmottnt of fluorescence corresponding to the amount of double-stranded nucleic acid has decreased about 70%.

A method of for assaying target polynucleotides in peripheral blood or bone marrow cells, said method comprising the steps of:

• depositing a specimen of blood or bone marrow on a solid support;
• contacting a cellular specirnen with a fixation medium to yield a fixed specimen having therein cells with substantially intact membranes, said fixation medium comprising at least one agent that is a precipitating agent or a cross-linking agent, said agent being an agent that allows double-stranded cellular polynucleotides to shift unnder hybridizing conditions to about 70% single-stranded cellular polynucleotides while maintaining cellular spatial relationships;
• incubating said fixed specimen with a hybridization solution comprising about 20% to 80% formnmide, about 5 times concentrated SSC, about 0.1M tris(hydroxymethyl)aminomethane-HCl , pH 7.4, about 0.1% of octylphenoxypolyethoxyethanol and a photobiotinylated single-stranded anti-sense RNA probe having 75-150 bases at least substantially complementary to a specific target nucleotide sequence to be detected said incubating being under hybridizing conditions so that double-strained cellular polynucleotides shift to about 70% single-stainded cellular polynucleotide, and
• adding an agent selected from avidin and streptavidin bearing a detectable label, said agent being at a concentration sufficient to bind said agent to said hybridized probe within a at least five minutes to form a probe-bound agent;
• washing said labeled specimen with a solution containing 0.1% octylphenoxypolyethoxyethanol to remove unbound agent;
• and
• detecing probe-bound agent in said cells with substantially intact membranes;
• wherein said shift under hybridizing conditions to about 70% single-stranded cellular polynucleotides while maintaining cellular spatial relationships is determinable by a process comprising the following four steps:
• (a) staining cells with a stain able to stain polynucleotides within said cells to yield a different emission spectrum upon ultraviolet excitation of stained double-straned and stained single-stranded polynucleotides;
• (b) determing the total amount of emitted fluorescence of a cell sample not treated under hybridizing conditions;
• (c) determining the total amount of emitted fluorescence of cell samples treated by various fixation and hybridization procedures and times;
• (d) assessing when the amount of fluorescence corresponding to the amount of double-stranded polynucleotides has decreased about 70%.

A method for assaying a target polynucleotide in tissue specimens, said method comprising the steps of:

• depositing a tissue specimen on a solid support;
• contacting said tissue specimen with a fixation medium to yield a fixed specimen having therein cells with substantially intact membranes while maintaining cellular spatial relationships;
• wherein said contacting is performed for at least 20 minutes at a temperature from -20.degree. C. to 50.degree. C., wherein said fixation medium comprises 50% methanol/50% acetone, so as to allow about a 70% shift of double-stranded to single-stranded cellular polynucleotides;
• incubating said fixed specimen with a hybridization solution comprising about 20% to 80% formamide, about 5 times concentrated SSC, about 0.1M sodium phosphate, pH 7.4, about 0.1% of octylphenoxypolyethoxyethanol, 20 mM vanadyl ribonucleoside complexes, a photobiotinylated single-stranded anti-sense RNA probe having 75-160 bases at least substantially complementary to a specific target nucleotide sequence to be detected, and low molecular weight denatured DNA at a concentration 100 times greater than the probe concentration said incubating being under hybridizing conditions so that the double-stranded cellular polynucleotides have shifted to about 70% single-strandedness, and
• adding a detectably labeled agent selected from avidin and streptavidin at a concentration sufficient to bind to duplexes formed between said probe and said target polynucleotide within at least 5 min to form bound detectably labeled agent;
• washing said labeled specimen with a solution containing 0.1% octylphenoxypolyethoxyethanol to remove unbound detectably labeled agent;
• and
• detecting said bound detectably labeled agent in said cells having substantially intact membranes, wherein:
• said shift under hybridizing conditions to about 70% single-stranded cellular polyucleotides while maiutnining cellular spatial relationships is determinable by a process comprising the following four steps:
• (a) staining cells with a stain able to stain polynucleotides within said cells to yield a different emission spectrum upon ultraviolet excitation of stained double-stranded and stained single-stranded polvnucleotides;
• (b) determining the total amount of emitted fluorescence of a cell sample not treated under hybridizing conditions;
• (c) determining the total mount of emitted fluorescence of cell snmples treated by various fixation and hybridization procedures and times;
• (d) assessing when the amount of fluorescence corresponding to the amount of double-stranded nucleic acid has decreased about 70%.

A method for selection of a fixative and determination of the optimum hybridization conditions for conducting an assay for a target polynucleotide in a cellular specimen, said assay comprising the steps of:

• (A) contacting a cellular specimen with a fixation medium to yield a fixed specimen having therein cells with substantially intact membranes, said fixation medium compriagent or a cross-linking agent, said precpitating agent or a cross-linking agent, said agent being an agent that allows double-stranded cellular polynucleotides to shift under hybridizing conditions to about 70% single-stranded cellular polynucleotides while maintaining cellular spatial relationships;
• (B) incubating said fixed specimen with a hybriclization solution comprising a denaturing agent, a hybrid stabilizing agent, a buffering agent, a membrane pore-forming agent and at least one probe having a nucleotide sequence at least substantially complementary to a specific target nucleotide sequence to be detected, said incubating being under hybridizing conditions so that double-stranded cellular polynucleotides skift to about 70% single-stranded cellular polynucleotides, and
• (C) detecting duplexes formed between said probe and said specific target nucleotide sequence in said cells with substantially intact membranes;
• wherein said method is capable of detecting as few as five polynucleotide molecules per cell;
• wherein selection of a fixstive comprises the steps of:
• (a) harvesting cells from a specimen;
• (b) applying a fixative;
• (c) incubating said cells with said fixative;
• (d) comparing the degree of preservation of cellular morphology after fixation to the morphology prior to fixation;
• (e) rating the fixation according to the retention of visual morphology;
• (f) rating the fixation according to the preservation of cellular antigens;
• and
• (g) selecting a fixative which scores satisfactorily in terms of preservation of cellular morphology and/or preservation of cellular antigenicity, and
• wherein determination of the optimum hybridization conditions comprises the steps of:
• (h) contacting cells from a specimen with said fixative selected in step (g);
• (i) incubating said fixed specimen with said hybridization solution, said contacting being under hybridizing conditions at a temperature from 15 to 80 degrees Celsius;
• (j) determining the shift of double-strandedness to single-strandedness by the cellular polynucleotides in the cells after step (i) has been performed at a desired temperature for a plurality of lengths of time by a process comprising the following four steps:
• (I) staining cells with a stain able to stain polynucleotides within said cells to yield a different emission spectrum upon ultraviolet excitation of stained double-stranded and stained single-stranded polynucleotides;
• (II) determining the total amount of emitted fluorescence of a cell sample not treated under hybridizing conditions;
• (III) determining the total amount of emitted fluorescence of cell samples treated by various fixation and hybridization procedures and times;
• (IV) assessing when the svnount of fluorescence corresponding to the amnount of double-stranded nucleic acid has decreased about 70%;
• and
• (k) Selecting the time for the incubating step (B) of the assay method as the time when about 70% of said cellular polynucleotide has shifted to single-stranded in step (j) and selecting the fixation medium of step (A) as the fixative selected in step (g) or, if about 70% of said cellular polynucleotide has not shifted to single-stranded after a desired length of time in step (j), selecting a different fixative in step (g) and performing steps (h) through (k) again.