US5514545A
Expired / Lapsed
US
⏱️ 0.0 years remaining
Method for characterizing single cells based on RNA amplification for diagnostics and therapeutics
Assignee: University of Pennsylvania
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Filing: 1993-07-26
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Granted: 1996-05-07
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Expires: 2013-07-26
Abstract
Abstract A method is provided for characterizing cells at the molecular level by amplifying RNA from selected single cells by microinjecting primer, nucleotides and enzyme into acutely dissociated cells to produce amplified antisense RNA; reamplifying the amplified antisense RNA (aRNA) produced by using random hexanucleotides to prime cDNA synthesis from aRNA, and then detecting messages in the amplified RNA. The invention is useful for characterization of cell identity or physiological state.
Inventors
- 👤 James Eberwine
Bibliographic Details
Application Number
—
Jurisdiction
US
Data Source
google_patents
Last Synchronized
2026-10-29 15:49
CPC Classifications
C; C12; C12Q; C12Q1/00; C12Q1/68; C12Q1/6844; C12Q1/6865
Claims Analysis
Total Claims: 7
Claim 1
Independent Claim
143 words
A method for characterizing mRNA in a single cell comprising the steps of: amplifying RNA in a single cell by microinjecting into a single cell an amplification primer, reverse transcriptase and nucleotides to synthesize a first strand cDNA from cellular mRNA; harvesting the cellular contents, including the first strand cDNA; synthesizing double-stranded cDNA from the first strand cDNA; and producing aRNA from the double stranded cDNA by addition of an RNA polymerase capable of recognizing the amplification primer; reamplifying the produced aRNA by adding random hexanucleotide primers; extending the hexanucleotide primers with a reverse transcriptase to form first strand cDNA; synthesizing double stranded cDNA from the first strand cDNA using the amplification primer; and producing reamplified aRNA from the double stranded cDNA by addition of an RNA polymerase capable of recognizing the amplification primer; and detecting specific RNA molecules in the reamplified aRNA.
Claim 2
Independent Claim
143 words
A method for characterizing mRNA in a single cell comprising the steps of: amplifying RNA in a single cell by microinjecting into a single cell an amplification primer, reverse transcriptase and nucleotides to synthesize a first strand cDNA from cellular mRNA; harvesting the cellular contents, including the first strand cDNA; synthesizing double-stranded cDNA from the first strand cDNA; and producing aRNA from the double stranded cDNA by addition of an RNA polymerase capable of recognizing the amplification primer; reamplifying the produced aRNA by adding random hexanucleotide primers; extending the hexanucleotide primers with a reverse transcriptase to form first strand cDNA; synthesizing double stranded cDNA from the first strand cDNA using the amplification primer; and producing reamplified aRNA from the double stranded cDNA by addition of an RNA polymerase capable of recognizing the amplification primer; and detecting specific RNA molecules in the reamplified aRNA.
Claim 3
Dependent Claim
16 words
The method of claim 1 wherein the amplification primer comprises oligo-dt-T7, oligo-dt-T3 or oligo-dT-SP 6 .
Claim 4
Dependent Claim
15 words
The method of claim 1 wherein the cell is a live acutely dissociated brain cell.
Claim 5
Dependent Claim
21 words
The method of claim 1 wherein the cell is first treated with an opioid compound prior to having its mRNA characterized.
Claim 6
Dependent Claim
28 words
The method of claim 4 further comprising the step of comparing the specific RNA molecules in the reamplified RNA of the opioid treated cell with a nontreated cell.
Claim 7
Dependent Claim
16 words
The method of claim 1 wherein the specific mRNA molecules are detected by reverse Northern blotting.
References Cited (Backward Citations)
US5021335A
Cited
US5168038A
Cited
USPTO Annuity & Maintenance Windows
| Annuity Stage | Window Opens | Payment Deadline | Surcharge Period Until | Estimated Fee | Status |
|---|---|---|---|---|---|
| 3.5 Years (Year 4) | 1999-05-07 | 1999-11-06 | 2000-05-07 | $2000.00 | Window Closed / Lapsed |
| 7.5 Years (Year 8) | 2003-05-07 | 2003-11-06 | 2004-05-07 | $3760.00 | Window Closed / Lapsed |
| 11.5 Years (Year 12) | 2007-05-07 | 2007-11-06 | 2008-05-07 | $7700.00 | Window Closed / Lapsed |