Nucleic acid quantitation from tissue slides

Abstract

This invention provides methods of quantitating nucleic acids from problematic samples, such as aged samples, formalin fixed samples, paraffin embedded samples, samples with aneuploid cells, and cells with fragmented nucleic acids. Methods include techniques to efficiently solubilize the nucleic acids under non-denaturing conditions from preserved clinical samples without resort to organic extractions, to normalize cell counts regardless of aneuploidy, to access the fragmentation state of the nucleic acids, and to provide standard curves for degraded nucleic acid samples.

1 - 19 . (canceled) 
     
     
         20 . A method of determining a number of test cells, the method comprising:
 obtaining a reference nucleic acid sample from a known number of reference cells;   quantitating an amount of a ribosomal DNA in the reference sample;   providing a standard function for the reference cell number versus the reference ribosomal DNA quantity;   obtaining a test nucleic acid sample from test cells;   quantitating an amount of the ribosomal DNA in the test sample; and,   determining a test cell number based on the standard function and the quantity of test ribosomal DNA.   
     
     
         21 . The method of  claim 20 , wherein the test cell is selected from the group consisting of: a tumor cell, a cell line, cells on a microscope slide, FFPE cells, and a polyploid cell. 
     
     
         22 . The method of  claim 21 , wherein the tumor cell comprises a lung tumor cell or a colon tumor cell. 
     
     
         23 . The method of  claim 20 , wherein said obtaining the reference nucleic acid sample comprises nucleic acid extraction from cells having a substantially normal karyotype. 
     
     
         24 . The method of  claim 20 , wherein the ribosomal DNA is selected from the group consisting of: 18S rDNA, 5.8S rDNA and 28S rDNA. 
     
     
         25 . The method of  claim 20 , wherein said quantitating comprises a technique selected from the group consisting of: bDNA analysis; Southern blot analysis, polymerase chain reaction, and agarose gel electrophoresis. 
     
     
         26 . The method of  claim 20 , wherein said determining the cell number comprises a method selected from the group consisting of: inputting the test ribosomal DNA quantity into the standard function, inputting the test ribosomal DNA quantity into a formula comprising a ratio of cells to rDNA, and inputting the test rDNA value into a computer. 
     
     
         27 . The method of  claim 20 , further comprising normalizing a result of a test cell analysis using the determined test cell number. 
     
     
         28 . The method of  claim 20 , further comprising determining an efficiency of test nucleic acid extraction based on a known test cell number and the determined test cell number. 
     
     
         29 . A method of determining RNA copy numbers, the method comprising:
 determining a number of cells in a test sample;   providing a standard function for an RNA assay output versus a degraded in vitro RNA standard assay input;   determining an amount of a test RNA in the test sample by the RNA assay using the standard function; and,   determining the copy number of the RNA in the cells based on the number of cells and the determined amount of test RNA.   
     
     
         30 . The method of  claim 29 , wherein the test sample is selected from the group consisting of: a tumor cell, a cell line, cells from a microscope slide, clinical samples more than a year old, cells fixed with formalin, cells embedded in paraffin, and a aneuploid cell. 
     
     
         31 . The method of  claim 29 , wherein the RNA standard is degraded by a degradation source selected from the group consisting of: time of storage, high pH, low pH, shear stress, a nuclease, light, heat, and contact with formaldehyde. 
     
     
         32 . The method of  claim 29 , wherein the quantitative analysis comprises a method selected from the group consisting of: bDNA analysis, northern blot analysis, RT-polymerase chain reaction, spectrophotometry, fluorometry, and agarose gel electrophoresis. 
     
     
         33 . A method of determining a RNA copy number for cells in a FFPE sample, said method comprising:
 providing a standard function for cell number versus quantity of a repetitive DNA;   separating paraffin from the cells after incubation in a protease solution at 40° C. or more;   determining a number of cells represented in the protease solution based on the standard function and an amount of the repetitive DNA in the protease solution;   providing a standard function of RNA assay output versus a degraded IVT RNA standard assay input;   determining an amount of the RNA in the protease solution based on the degraded IVT RNA standard function; and,   calculating the RNA copy number per cell by dividing the determined number of cells by the determined amount of RNA.   
     
     
         34 . The method of  claim 33 , wherein the repetitive DNA comprises a ribosomal DNA. 
     
     
         35 . The method of  claim 33 , wherein the protease is a proteinase K and the incubation temperature is about 65° C. 
     
     
         36 . The method of  claim 33 , wherein the RNA assay comprises a bDNA assay, RT-PCR, or a northern blot. 
     
     
         37 . The method of  claim 33 , further comprising selecting the degraded IVT RNA standard or modifying a slope of a standard curve according to the results of an offset bDNA assay. 
     
     
         38 . A method of detecting fragmentation of a nucleic acid, the method comprising:
 analyzing a sample of the nucleic acid in an offset bDNA assay wherein each of one or more offset capture extender probe C3 sequences are complimentary to sequences along the nucleic acid, and wherein one or more offset label extender L1 sequences are complimentary to sequences of the nucleic acid spaced at least one nucleotide base either 5′ or 3′ from all the C3 complimentary sequences;   wherein less signal is generated in the bDNA assay if the nucleic acid is fragmented between the C3 and L1 complimentary sequences than if the nucleic acid is not fragmented between the C3 and L1 complimentary sequences.   
     
     
         39 . The method of  claim 38 , further comprising:
 analyzing the sample of the nucleic acid in a second bDNA assay wherein two or more control capture extender probe C3 sequences are complimentary to sequences at different positions along the nucleic acid, and wherein one or more control label extender L1 sequences are complimentary to sequences at different positions along the nucleic acid sequence; with one or more of the control L1 sequences being complimentary to the nucleic acid at positions between the positions complimentary to two or more of the control C3 sequences;   wherein, if the nucleic acid is fragmented, a ratio of a control assay result over the offset assay result is higher than the ratio if the nucleic acid is not fragmented.   
     
     
         40 . The method of  claim 38 , wherein the nucleic acid is fragmented. 
     
     
         41 . The method of  claim 38 , wherein the nucleic acid is selected from the group consisting of: an RNA, an mRNA, a cDNA, a nucleic acid from a FFPE sample, a nucleic acid that does not have intron sequences, and a nucleic acid from a cell or tissue sample more than a year old. 
     
     
         42 . The method of  claim 38 , wherein the nucleic acid sequences complimentary to the offset C3 sequences are separated from the nucleic acid sequences complimentary to the offset L1 sequences by a space of 75% or more of the nucleic acid nucleotides. 
     
     
         43 . The method of  claim 38 , wherein the one or more offset label extender L1 sequences are complimentary to sequences of the nucleic acid spaced at least 25 nucleotide bases either 5′ or 3′ from all the C3 complimentary sequences. 
     
     
         44 . The method of  claim 38 , wherein the one or more spacing nucleotides comprise a sequence complimentary to a blocking probe. 
     
     
         45 . The method of  claim 38 , wherein no L1 complimentary sequence is between any two C3 complimentary sequences. 
     
     
         46 . The method of  claim 39 , further comprising correlating the ratio to an average fragment length of the nucleic acid. 
     
     
         47 . The method of  claim 39 , further comprising selecting an assay standard or selecting a standard function based on the ratio.