Method of defining cell types by probing comprehensive expression libraries with amplified RNA

• US 6,110,711 A
• Filed: 12/15/1998
• Issued: 08/29/2000
• Est. Priority Date: 12/12/1997
• Status: Expired due to Fees

• Current Assignee: Regents of the University of California (University of California)
• Original Assignee: Regents of the University of California (University of California)
• Inventors: Serafini, Tito, Ngai, John
• Primary Examiner(s): Horlick, Kenneth R.
• Assistant Examiner(s): Siew, Jeffrey
• Application Number:
  US09/212,338
• Time in Patent Office: 623 Days
• Field of Search: 435/6, 435/91.2, 435/91.21, 536/23.1, 536/24.31, 536/24.33
• US Class Current: 435/91.21
• CPC Class Codes: C12N 15/1096 cDNA Synthesis; Subtracted cDNA library construction, e.g. RT, RT-PCR
• CPC Class Codes: C12N 2517/02 Cells from transgenic animals
• CPC Class Codes: C12Q 1/6809 Methods for determination or identification of nucleic acids involving differential detection
• CPC Class Codes: C12Q 1/6855 Ligating adaptors
• CPC Class Codes: C12Q 1/6865 Promoter-based amplification, e.g. nucleic acid sequence amplification [NASBA], self-sustained sequence replication [3SR] or transcription-based amplification system [TAS]
• CPC Class Codes: C12Q 1/6897 involving reporter genes operably linked to promoters
• CPC Class Codes: C12Q 2525/191 incorporating an adaptor
• CPC Class Codes: C12Q 2531/143 Promoter based amplification, e.g. NASBA, 3SR, TAS
• CPC Class Codes: C12Q 2565/518 characterised by the immobilisation of the nucleic acid sample or target

Abstract

The invention provides methods and compositions for defining a cell type, generally involving the steps of (a) amplifying the mRNA of a single cell of a heterogenous population of cells; (b) probing a comprehensive expression library with the amplified mRNA to define a gross expression profile of the cell; and (c) comparing the gross expression profile of the cell with a gross expression profile of one or more other cells to define a unique expression profile of the cell, wherein the unique expression profile of the cell provides a marker defining the cell type.

Claims

1. A method for defining a cell type of a cell comprising the steps of:

(a) amplifying the mRNA of a single cell, of undefined cell type of a heterogenous population of cells;

(b) probing a comprehensive expression library with the amplified mRNA to define a gross expression profile of the cell;

(c) comparing the gross expression profile of the cell with a gross expression profile of one or more other cells to define a unique expression profile of the cell,

wherein the unique expression profile of the cell provides a marker defining the cell type.

2. A method according to claim 1, wherein step (c) comprises comparing the gross expression profile of the cell with a gross expression profile of a plurality of other cells to define a unique expression profile of the cell.

3. A method according to claim 1, wherein step (c) comprises comparing the gross expression profile of the cell with a plurality of gross expression profiles of each of a plurality of other single cells to define a unique expression profile of the cell.

4. A method according to claim 1, wherein step (c) comprises comparing the gross expression profile of the cell with a plurality of gross expression profiles of each of a plurality of other single cells to define a unique expression profile of the cell, and the plurality of other single cells are derived from a functionally or structurally distinct subpopulation of cells.

5. A method according to claim 4, wherein the subpopulation of cells is tissue-specific.

6. A method according to claim 4, wherein each cell of the subpopulation of cells comprises a genome comprising a mutation.

7. A method according to claim 4, wherein each cell of the subpopulation of cells comprises a genome comprising a mutation, wherein the mutation provides each cell with a common selectable or detectable marker.

8. A method according to claim 4, wherein each cell of the subpopulation of cells comprises a genome comprising a mutation, wherein the mutation is an inserted construct which encodes and provides each cell with a common selectable marker selected from an epitope or a signal-producing protein.

9. A method according to claim 4, wherein each cell of the subpopulation of cells comprises a genome comprising a mutation, wherein the mutation is an inserted construct which encodes and provides each cell a signal-producing protein selected from the group consisting of a green fluorescent protein, a galactosidase and an externally accessible, cell-surface associated protein.

10. A method according to claim 4, wherein each cell of the subpopulation of cells comprises a genome comprising a mutation, wherein the mutation is an inserted construct which encodes and provides each cell with a common selectable marker selected from the group consisting of an epitope or a signal-producing protein, and the inserted construct further encodes and provides each cell an internal ribosome entry sequence and the construct is inserted into a target gene downstream of the stop codon but upstream of the polyadenylation signal in the last exon of the target gene, such that the internal ribosome entry sequence provides a second open reading frame within a transcript of the target gene.

11. A method according to claim 4, wherein each cell of the subpopulation of cells comprises a genome comprising a mutation, wherein the mutation is an inserted construct which encodes and provides each cell with a common selectable marker selected from an epitope or a signal-producing protein, and the subpopulation is isolated by flow cytometry.

12. A method according to claim 1, wherein the library is a cDNA library.

13. A method according to claim 1, wherein the library is normalized or subtracted.

14. A method according to claim 1, wherein the library comprises a high density ordered array of immobilized nucleic acids.

15. A method according to claim 1, wherein the mRNA is amplified by a method comprising the steps of adding a known nucleotide sequence to the 3'"'"' end of a first RNA having a predetermined sequence at the 5'"'"' end to form a second RNA and reverse transcribing the second RNA to form a cDNA.

16. A method according to claim 1, wherein the mRNA is amplified by a method comprising the steps of adding a predetermined nucleotide sequence to the 3'"'"' end of a first RNA having a predetermined sequence at the 5'"'"' end to form a second RNA and reverse transcribing the second RNA to form a cDNA and wherein the cDNA is single-stranded and converted to a double-stranded cDNA by a method comprising the steps of contacting the RNA with a denaturant and contacting the single-stranded cDNA with a DNA polymerase and an oligonucleotide primer comprising a sequence complementary to the 3'"'"' end of the single-stranded cDNA and an RNA polymerase promoter, whereby the DNA polymerase initiates the conversion at a noncovalently joined duplex region of the 3'"'"' end of the single-stranded cDNA and the oligonucleotide primer.

17. A method according to claim 1, wherein the mRNA is amplified by a method comprising the steps of adding a known predetermined nucleotide sequence to the 3'"'"' end of a first RNA having a predetermined sequence at the 5'"'"' end to form a second RNA and reverse transcribing the second RNA to form a cDNA and wherein the cDNA is single-stranded and converted to a double-stranded cDNA by a method comprising the steps of contacting the RNA with a denaturant and contacting the single-stranded cDNA with a DNA polymerase and an oligonucleotide primer comprising a sequence complementary to the 3'"'"' end of the single-stranded cDNA and an RNA polymerase promoter comprising SEQ ID NO:

1 joined to an upstream flanking sequence of about 3 to 100 nucleotides, whereby the DNA polymerase initiates the conversion at a noncovalently joined duplex region of the 3'"'"' end of the single-stranded cDNA and the oligonucleotide primer.

18. A method according to claim 1, wherein the mRNA is amplified by a method comprising the steps of adding a predetermined nucleotide sequence to the 3'"'"' end of a first RNA having a predetermined sequence at the 5'"'"' end to form a second RNA and reverse transcribing the second RNA to form a cDNA and wherein the mRNA is amplified by a method comprising the steps of adding a known nucleotide sequence to the 3'"'"' end of a first RNA having a known sequence at the 5'"'"' end to form a second RNA and reverse transcribing the second RNA to form a cDNA and wherein the cDNA is single-stranded and converted to a double-stranded cDNA, and the method further comprises the step of repeatedly transcribing the double-stranded cDNA to form a plurality of third RNAs.

19. A method according to claim 1, wherein the mRNA is amplified by a method comprising the steps of adding a determined nucleotide sequence to the 3'"'"' end of a first RNA having a predetermined sequence at the 5'"'"' end to form a second RNA and reverse transcribing the second RNA to form a cDNA and wherein the first RNA is made by amplifying a mRNA.

20. A method according to claim 1, wherein the mRNA is amplified by a method comprising the steps of adding a predetermined nucleotide sequence to the 3'"'"' end of a first RNA having a predetermined sequence at the 5'"'"' end to form a second RNA and reverse transcribing the second RNA to form a cDNA, wherein the first RNA is made by amplifying a mRNA by the steps of hybridizing to the poly(A) tail of the mRNA a poly(T) oligonucleotide joined to an RNA polymerase promoter sequence, reverse transcribing the mRNA to form single-stranded cDNA, converting the single-stranded cDNA to a double-stranded cDNA and transcribing the double-stranded cDNA to form the first RNA.

21. A method according to claim 1, wherein the mRNA is amplified by a method comprising a polymerase chain reaction.