siRNA DETECTION METHOD
AssigneeGenecare Research Institute Co., Ltd.
InventorsKazunobu Futami, Yasuhiro Furuichi
Life of this patent
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Abstract
PolydG is added to the terminal overhangs of an siRNA. Next, a primer containing a polydC sequence added with a tag sequence is annealed and cDNA is synthesized by a reverse transcription reaction. Quantitative PCR is performed between a primer carrying the same sequence as the tag sequence, and a primer containing the same sequence as the siRNA sequence to be detected. The amount of siRNA of interest can be determined from a calibration curve produced using known amounts of short-chain dsDNA.
Claims7
A method for detecting an siRNA comprising a DNA overhang at the 3′ end, which comprises the steps of:
- (a) adding polydeoxyguanine (polydG) to the overhang of an siRNA;
- (b) synthesizing a single-stranded DNA by performing a reverse transcription reaction using the molecule produced by the previous step as a template and a polydeoxycytosine (polydC) primer comprising a tag sequence at the 5′ side;
- (c) performing PCR using the single-stranded DNA as a template, a primer that anneals to the complementary strand of the tag sequence, and a primer that anneals to one of the strands of the siRNA;
- and
- (d) detecting the PCR product.
A method for selectively amplifying a sequence constituting an siRNA comprising a DNA overhang at the 3′ end, which comprises the steps of:
- (a) adding polydeoxyguanine (polydG) to the overhang of an siRNA;
- (b) synthesizing a single-stranded DNA by performing a reverse transcription reaction using the molecule produced by the previous step as a template and a polydeoxycytosine (polydC) primer comprising a tag sequence at the 5′ side;
- and
- (c) performing PCR using the single-stranded DNA as a template, a primer that anneals to the complementary strand of the tag sequence, and a primer that anneals to one of the strands of the siRNA.
A reagent for detecting an siRNA comprising a DNA overhang at the 3′ end, which comprises:
- a polydeoxycytosine (polydC) primer comprising a tag sequence at the 5′ side;
- a primer that anneals to a strand complementary to the tag sequence;
- or
- a primer that anneals to one of the strands of the siRNA.
A method for detecting an siRNA comprising a DNA overhang at the 3′ end, which comprises the steps of:
- (a) adding polydeoxyguanine (polydG) to the overhang of an siRNA;
- (b) synthesizing a single-stranded DNA by performing a reverse transcription reaction using the molecule produced by the previous step as a template and a polydeoxycytosine (polydC) primer comprising a tag sequence at the 5′ side;
- (c) performing PCR using the single-stranded DNA as a template, the primer, and a primer that anneals to one of the strands of the siRNA;
- and
- (d) detecting the PCR product.
A method for selectively amplifying a sequence constituting an siRNA comprising a DNA overhang at the 3′ end, which comprises the steps of:
- (a) adding polydeoxyguanine (polydG) to the overhang of an siRNA:
- (b) synthesizing a single-stranded DNA by performing a reverse transcription reaction using the molecule produced by the previous step as a template and a polydeoxycytosine (polydC) primer comprising a tag sequence at the 5′ side;
- and
- (c) performing PCR using the single-stranded DNA as a template, the primer, and a primer that anneals to one of the strands of the siRNA.
Dependencies are inferred from the claim text.