Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
1999-11-24
2001-06-19
Zitomer, Stephanie (Department: 1655)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S091100, C435S091200, C435S091210, C536S023100, C536S023200, C536S023500, C536S024300, C536S024310, C536S024320, C536S024330
Reexamination Certificate
active
06248534
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates to a method of detecting RNA and in particular to a method of detecting RNA, in which RNA can be detected highly accurately.
2. Description of the Related Art
In order to determine the level of gene expression, generally, Northern hybridization is carried out. For the routine determination at the laboratory level, the presence of 5 pg (picograms) of RNA is sufficient for detection. However, in cases where the amount of expression of a target gene is extremely small, 0.3-3 &mgr;g of mRNA is required for detection. Thus, it is difficult to apply Northern hybridization to cases where samples of only limited amounts are available (e.g., clinical samples).
Polymerase chain reaction (PCR) is a technique by which DNA or RNA can be detected most sensitively compared to other techniques. However, quantitative determination of gene expression by PCR involves a control experiment in which a calibration curve is prepared using, as the so-called “internal control”, a DNA fragment having an amplification efficiency similar to that of a target molecule. Thus, operations are complicated. Furthermore, in order to perform quantitative PCR with a number of genes, it is necessary to prepare a calibration curve for each of the target genes to be quantitatively determined. Thus, a study of genes or a genetic diagnosis with this method requires much time and labor.
SUMMARY OF THE INVENTION
It is an object of the present invention to provide (1) a method of detecting RNA, (2) a method of detecting RNA in which RNA can be detected highly accurately, and (3) a method of detecting RNA in which a small quantified amount of a sample can also be detected and a number of samples can be easily quantified.
As a result of intensive and extensive research toward the solution of the problems described above, the present inventors have found that a target RNA can be detected highly accurately by adding a different adapter to each of RNA-derived cDNA as the subject of detection and cDNAs used for preparing a. standard curve, and then performing PCR. Thus, the present invention has been achieved.
That is, the present invention relates to a method of detecting RNA, comprising:
(a) preparing cDNA to be measured and standard cDNA by reverse transcription of RNA as the subject of detection and standard RNA respectively,
(b) adding different adapters respectively to the standard cDNA prepared at stepwise concentrations,
(c) adding an adapter other than said adapters to the cDNA to be measured,
(d) mixing and amplifying the adapter-tagged cDNAs obtained in the steps (b) and (c),
(e) measuring the ratio of the a target cDNA to the amount of standard cDNA, and
(f) detecting said RNA from the measurement results.
The different adapters include those containing nucleotides which are different in length from one another. The amplification includes that using an adapter primer and a gene-specific primer.
REFERENCES:
patent: 6090556 (2000-07-01), Kato
patent: 10337200 (1988-12-01), None
Kato K. Adapter-tagged competitive PCR: A novel method for measuring relative gene expression. Nucleic acids Research, vol. 25, No. 22, pp. 4694-4696, Nov. 1997.*
The 21st Annual Meeting of the Molecular Biology Society of Japan Dec. 16 to Dec. 19, 1998, Abstract of Paper P-547 “Mutiplex Adaptor-Tag Competitive PCR,” Ryo Matoba et al.
Kato Kikuya
Matoba Ryou
Kato Kikuya
Lorusso & Loud
Wilder Cynthia B.
Zitomer Stephanie
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