Methods of measuring gene expression using wax-embedded tissue specimens

• US 6,183,995 B1
• Filed: 09/05/1997
• Issued: 02/06/2001
• Est. Priority Date: 09/05/1997
• Status: Expired due to Fees

• Current Assignee: LifeSpan Biosciences Incorporated
• Original Assignee: LifeSpan Biosciences Incorporated
• Inventors: Burmer, Glenna C., Brown, Joseph P., Ford, Amanda A.
• Primary Examiner(s): Achutamurthy, Ponnathapu
• Assistant Examiner(s): Moore, William W.
• Application Number:
  US08/925,818
• Time in Patent Office: 1,250 Days
• Field of Search: 435/91.1, 435/91.2, 435/91.5, 435/91.51, 435/6, 436/503, 436/94, 436/503.94, 536/23.1, 536/24.2, 536/24.3, 536/24.33, 536/25.32, 935/77, 935/78, 935/86, 935/87, 422/68.1
• US Class Current: 435/91.1
• CPC Class Codes: C12N 15/1003 Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor
• CPC Class Codes: C12N 15/1096 cDNA Synthesis; Subtracted cDNA library construction, e.g. RT, RT-PCR
• CPC Class Codes: C12Q 1/6806 Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay C12Q1/6804 takes precedence
• CPC Class Codes: C12Q 1/6809 Methods for determination or identification of nucleic acids involving differential detection
• CPC Class Codes: Y10T 436/143333 Saccharide [e.g., DNA, etc.]

Abstract

This invention provides methods for the isolation of complete representation of cellular messenger ribonucleic acid from wax-embedded tissue samples. Expression of genes of interest can thus be fortuitously determined and cDNA probes can be readily developed.

Claims

1. A method for the production of cDNA representative of complete representational cellular mRNA from a wax-embedded biological sample comprising:

(a) extracting RNA from the wax-embedded sample by removing the DNA in the biological sample with a DNAse and without a step to selectively eliminate rRNA by selection for polyadenylated RNA to produce sample cellular RNA; and

then

(b) synthesizing a first strand of cDNA complementary to the sample cellular mRNA by contacting the sample RNA to one or more oligonucleotides and a reverse transcriptase to create single strand cDNA copies of the cellular RNA representative of the complete cellular mRNA; and

(c) ligating a pair of complementary oligonucleotide adaptors to the cDNA wherein the adaptors are of different lengths.

2. The method of claim 1, comprising further a step of synthesizing complementary DNA strands to the cDNA copies to create double-stranded cDNA after step (b).

3. The method of claim 1, comprising further a step of amplifying the cDNA copies with a primer complementary to one of the adaptors.

4. The method of claim 3, wherein the amplified cDNA copies are separated from one another by size or sequence specificity.

5. The method of claim 4, wherein the cDNA probes are produced directly by cDNA synthesis with labeled oligonucleotides or dNTPs.

6. The method of claim 1, wherein one of the adaptors in the pair is about 6 to 18 nucleotides in length and shorter than the other adaptor.

7. The method of claim 1, wherein the cDNA is synthesized using labeled oligonucleotides or labeled dNTPs.

8. The method of claim 1, wherein the first adaptor and the second adaptor contain a sequence for a restriction endonuclease site.

9. The method of claim 1, wherein the wax-embedded sample is formalin-fixed.

10. The method of claim 1, wherein the biological sample is a human sample and the adaptors are not homologous to a nucleic acid sequence found in a human genome.

13. A method for determining the presence or absence of gene expression in a wax-embedded biological sample comprising, extracting mRNA from wax-embedded biological material;

synthesizing at least two cDNA probes from the wax-embedded biological material obtained pursuant to the method of claim 2 and screening the probes against 1 nucleic acid material containing a known gene of interest to determine if the cDNA probes hybridize with the gene of interest.

14. A method for comparing the genes in two separate biological sources by contacting a known nucleic acid probe representative of one source with an array of cDNA obtained in accordance with the method of claim 2 under conditions suitable for hybridization and determining whether hybridization of the probe to a cDNA fragment occurs.

11. A method for the production of cDNA representative of complete representational cellular mRNA from a wax-embedded biological sample comprising:

(a) extracting RNA from the wax-embedded sample by removing the DNA in the biological sample with a DNAse and without a step to selectively eliminate rRNA by selection for polyadenylated RNA to produce sample cellular RNA;

(b) synthesizing a first strand of cDNA complementary to the sample cellular mRNA by contacting the sample RNA to one or more oligonucleotides and a reverse transcriptase to create single strand cDNA copies of the cellular RNA representative of the complete cellular mRNA;

(c) synthesizing complementary DNA strands to the cDNA copies to create double-stranded cDNA;

(d) ligating a pair of complementary oligonucleotide adaptors to the cDNA wherein the adaptors are of different lengths; and

wherein one of the adaptors in the pair is about 6 to 18 nucleotides in length and shorter than the other adaptor.

12. A method for the production of cDNA representative of complete representational cellular mRNA from a wax-embedded biological sample comprising:

(a) extracting RNA from the wax-embedded sample by removing the DNA in the biological sample with a DNAse and without a step to selectively eliminate rRNA by selection for polyadenylated RNA to produce sample cellular RNA;

(b) synthesizing a first strand of cDNA complementary to the sample cellular mRNA by contacting the sample RNA to one or more oligonucleotides and a reverse transcriptase to create single strand cDNA copies of the cellular RNA representative of the complete cellular mRNA;

(c) synthesizing complementary DNA strands to the cDNA copies to create double-stranded cDNA; and

(d) ligating a pair of complementary oligonucleotide adaptors to the cDNA wherein the adaptors are of different lengths and arc not homologous to DNA in the biological sample.

15. A method for comparing and quantifying expression of at least three genes of interest in a wax-embedded biological source comprising,

(a) extracting mRNA from a wax-embedded biological source to obtain complete representational cellular mRNA present in the source;

(b) synthesizing cDNA probes corresponding to the complete representational RNA; and

(c) contacting the cDNA probes with DNA representative of at least three different genes and detecting the proportionate copy number of the cDNA probes complementary to DNA from the different genes.

16. The method of claim 15, further wherein steps (a)-(c) are repeated with multiple sources % mRNA.