Methods of detecting nucleic acids in individual cells and of identifying rare cells from large heterogeneous cell populations

Abstract

Methods of detecting multiple nucleic acid targets in single cells through indirect capture of labels to the nucleic acids are provided. Methods of assaying the relative levels of nucleic acid targets through normalization to levels of reference nucleic acids are also provided. Methods of detecting individual cells, particularly rare cells from large heterogeneous cell populations, through detection of nucleic acids are described. Related compositions, systems, and kits are also provided.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS[0001]This application is a non-provisional utility patent application claiming priority to and benefit of the following prior provisional patent application: U.S. Ser. No. 60 / 691,834, filed Jun. 20, 2005, entitled “Method of Detecting and Enumerating Rare Cells from Large Heterogeneous Cell Populations” by Luo and Chen, which is incorporated herein by reference in its entirety for all purposes.FIELD OF THE INVENTION[0002]The invention relates generally to nucleic acid chemistry and biochemical assays. More particularly, the invention relates to methods for in situ detection of nucleic acid analytes in single cells. The invention also relates to detection and identification of single cells, particularly rare cells.BACKGROUND OF THE INVENTION[0003]Ample evidence has demonstrated that cancer cells can dissociate from the primary tumor and circulate in the lymph node, bone marrow, peripheral blood or other body fluids. These circulating tumor cell...

AI Technical Summary

Benefits of technology

[0041]As another example, the third nucleic acid target can serve as a third redundant marker for the target cell type, e.g., to improve specificity of the assay for the desired cell type. Thus, in one class of embodiments, the methods include correlating the third signal detected from the cell with the presence, absence, or amount of the third nucleic acid target in the cell, and identifying the cell as being of the specified type based on detection of the presence, absence, or amount of the first, second, and third nucleic acid targets within the cell, wherein the specified type of cell is distinguishable from the other cell type(s) in the mixture on the basis of either presence, absence, or amount of the first nucleic acid target, presence, absence, or amount of the second nucleic acid target, or presence, absence, or amount of the third nucleic acid target in the cell.

Problems solved by technology

Validation of the clinical utility of CTC detection as a prognostic indicator has not been progressing as fast as expected, in large part due to lack of suitable detection technologies.

One key difficulty in detecting CTC in peripheral blood or other body fluids is that CTC are present in the circulation in extremely low concentrations, estimated to be in the range of one tumor cell among 106-107 normal white blood cells.

Although this technology has reported high sensitivity, its applicability is limited by the availability of detection antibodies that are highly sensitive and specific to particular types of CTC.

The antibodies can exhibit non-specific binding to other cellular components which can lead to low signal to noise ratio and impair later detection.

The antibodies binding to CTC may also bind to antigen present in other types of cells at low level, resulting in a high level of false positives.

These results suggest that tumor cells were shed into the bloodstream and resulted in poor patient outcomes in patients with colorectal cancer.

However, the QPCR approach requires the laborious procedure of mRNA isolation from the blood sample and reverse transcription before the PCR reaction.

False positives are often observed using this technique due to sample contamination by chromosomal DNA or low-level expression of the chosen marker gene in normal blood cells (Fava et al.

In addition, the limit of detection sensitivity of this technique is at most about one tumor cell per 1 ml of blood, and the technology cannot provide an accurate count of CTC numbers.

Currently available techniques do not fulfill these needs.

However, ISH technology faces a number of technical challenges that limit its wide use.

First of all, cells immobilized on solid surface exhibit poor hybridization kinetics.

In addition to technical issues, current ISH technology has relatively low performance standards in term of its detection sensitivity and reproducibility.

The false positive rate is still high unless the relevant cells are re-examined manually using their morphology, which is time and labor-intensive.

Current ISH technology also does not have the capability to quantitatively determine the mRNA expression level or to simultaneously measure the expression of multiple target mRNA within cells, which may provide clinical valuable information such as increased detection sensitivity and specificity, and the identification of primary tumor type, source and stage.

RNA probe is a direct labeling method that suffers a number of difficulties.

Second, it is technically difficult to detect the expression of multiple mRNA of interest in situ at the same time.

Furthermore, with direct labeling methods, there is no good way to control for potential cross-hybridization with non-specific sequences in cells.

The nonspecific hybridization of the oligonucleotide probes in bDNA ISH can become a serious problem when multiple of those probes have to be used for the detection of low abundance mRNAs.

Similarly, although use of bDNA ISH to detect or quantitate multiple mRNAs is desirable, such nonspecific hybridization of the oligonucleotide probes is a potential problem.

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