Back to results
US 12,767,987 B2Granted
Method for screening multiplex reverse transcription primer combinations for quantifying multiple mirnas
AssigneeMiracle Biotechnology Inc.
InventorsChang Su, Yuan Cao, Mengjing Cao, Demei Ma
Life of this patent
Estimating the term…
Abstract
A method for selecting and screening a multiplex reverse transcription primer combination for quantifying multiple target miRNAs simultaneously, and the application of the multiplex reverse transcription primer combination for determining a physiological or mental condition of a living subject.
Claims17
A method for screening a multiplex stem-loop reverse transcription primer combination demonstrating multi-specificity for quantifying a plurality of target miRNAs simultaneously, the method comprising:
- (A) providing the plurality of target miRNAs of a human subject, wherein each of the plurality of target miRNAs associated with the health condition has a unique 3′ sequence;
- wherein each of the plurality of target miRNAs has a length between about 18-25 nt;
- and the plurality of target miRNAs includes 2-10 target miRNAs;
- (B) providing a plurality of stem-loop reverse transcription primers, wherein each of the plurality of stem-loop reverse transcription primers has a stem-loop sequence forming a stem-loop structure and an anchor sequence complimentary to the unique 3′ sequence of one of the plurality of target miRNAs,
- wherein the plurality of stem-loop reverse transcription primers are selected by a selection process comprising:
- a) selecting the stem-loop sequence for each of the plurality of stem-loop reverse transcription primers;
- and
- b) selecting a certain length for the anchor sequence of each of the plurality of stem-loop reverse transcription primers,
- wherein the length of the anchor sequence is selected between 3 nt to 12 nt;
- and
- wherein the lengths of the anchor sequences of each of the plurality of stem-loop reverse transcription primers is different from the others;
- (C) screening the plurality of stem-loop reverse transcription primers by a screening process for determining the multi-specificity of the plurality of stem-loop reverse transcription primers, wherein the screening process comprises:
- i) separately performing a process of singleplex reverse transcription for each of the plurality of stem-loop reverse transcription primers using a mixed target miRNAs template comprising the plurality of target miRNAs to be quantified;
- ii) separately collecting a collection of reverse transcription products from each of the separate singleplex reverse transcription processes of step i);
- iii) separately performing a process of multiplex qPCR amplification for each of the collections of reverse transcription products from step ii) to form a collection of multiplex qPCR products for each of the collections of reverse transcription products;
- iv) separately obtaining a quantification result of each of the plurality of target miRNAs based on each of the collections of multiplex qPCR products and obtaining a collection of the quantification results of the plurality of target miRNAs;
- and
- v) determining the multi-specificity of the plurality of stem-loop reverse transcription primers based on the collection of quantification results formed in step iv), wherein the multi-specificity of the plurality of stem-loop reverse transcription primers is confirmed when the collection of quantification results shows that each of the plurality of stem-loop reverse transcription primers specifically reverse transcribes the target miRNA to which the anchor sequence of each of the plurality of stem-loop reverse transcription primer is complimentary;
- and
- (D) modifying, if the multi-specificity of the plurality of stem-loop reverse transcription primers is not confirmed by the collection of quantification results, the plurality of stem-loop reverse transcription primers by a repeat process comprising:
- providing a modified plurality of stem-loop reverse transcription primers by repeating step b) of the selection process to select a new length for the anchor sequence of each of those of the plurality of stem-loop reverse transcription primers which fail to specifically reverse transcribe the target miRNA to which the anchor sequence of each of the plurality of stem-loop reverse transcription primers is complimentary, wherein the length of the anchor sequence is selected between 3 nt to 12 nt;
- wherein, after the modification, the lengths of the anchor sequences of each of the modified plurality of stem-loop reverse transcription primers is different from the others;
- and
- repeating steps i)-v) of the screening process to determine the multi-specificity of the modified plurality of stem-loop reverse transcription primers, until the modified plurality of stem-loop reverse transcription primers demonstrates the multi-specificity and quantifies the plurality of target miRNAs simultaneously.
21 more elements
A method for screening a multiplex reverse transcription primer combination demonstrating multi-specificity for quantifying a plurality of target miRNAs of a human subject simultaneously, the method comprising:
- providing the plurality of target miRNAs, wherein each of the plurality of target miRNAs has a unique 3′ sequence;
- wherein each of the plurality of target miRNAs has a length between about 18-25 nt;
- and the plurality of target miRNAs includes 2-10 target miRNAs;
- providing a plurality of stem-loop reverse transcription primers, wherein each of the plurality of stem-loop reverse transcription primers has a stem-loop sequence forming a stem-loop structure and an anchor sequence complimentary to the unique 3′ sequence of one of the plurality of target miRNAs,
- wherein the plurality of stem-loop reverse transcription primers are selected by a selection process comprising:
- a) selecting the stem-loop sequence for each of the plurality of stem-loop reverse transcription primers;
- and
- b) selecting a certain length for the anchor sequence of each of the plurality of stem-loop reverse transcription primers,
- wherein the lengths of the anchor sequences of each of the plurality of stem-loop reverse transcription primers is different from the others;
- screening the plurality of stem-loop reverse transcription primers by a screening process for determining the multi-specificity of the plurality of stem-loop reverse transcription primers;
- wherein the screening process comprises:
- separately performing a process of singleplex reverse transcription for each of the plurality of stem-loop reverse transcription primers using a mixed target miRNAs template comprising the plurality of target miRNAs to be quantified;
- separately collecting a collection of reverse transcription products from each of the separate singleplex reverse transcription processes;
- separately performing a process of multiplex qPCR amplification for each of the collections of reverse transcription products to form a collection of multiplex qPCR products for each of the collections of reverse transcription products;
- separately obtaining a quantification result of each of the plurality of target miRNAs based on each of the collections of multiplex qPCR products and obtaining a set of the quantification results of the plurality of target miRNAs;
- and
- determining the multi-specificity of the plurality of stem-loop reverse transcription primers based on the set of quantification results, wherein the multi-specificity of the plurality of stem-loop reverse transcription primers is confirmed when the set of quantification results shows that each of the plurality of stem-loop reverse transcription primers specifically reverse transcribes the target miRNA to which the anchor sequence of each of the plurality of stem-loop reverse transcription primer is complimentary.
14 more elements
A method for screening a multiplex stem-loop reverse transcription primer combination demonstrating multi-specificity for quantifying a plurality of target miRNAs of a human subject simultaneously, comprising the steps of:
- (A) providing the plurality of target miRNAs wherein each of the plurality of target miRNAs has a unique 3′ sequence;
- wherein each of the plurality of target miRNAs has a length between about 18-25 nt;
- and the plurality of target miRNAs includes 2-10 target miRNAs;
- (B) providing a plurality of stem-loop reverse transcription primers, wherein each of the plurality of stem-loop reverse transcription primers has a stem-loop sequence forming a stem-loop structure and an anchor sequence complimentary to the unique 3′ sequence of one of the plurality of target miRNAs, and wherein the lengths of the anchor sequences of at least two of the plurality of stem-loop reverse transcription primers are different from each other;
- (C) screening the plurality of stem-loop reverse transcription primers to determine the multi-specificity of the plurality of stem-loop reverse transcription primers, wherein the multi-specificity of the plurality of stem-loop reverse transcription primers is confirmed when each of the plurality of stem-loop reverse transcription primers specifically reverse transcribes the target miRNA to which the anchor sequence of each of the plurality of stem-loop reverse transcription primer is complimentary;
- and
- (D) modifying, if the multi-specificity of the plurality of stem-loop reverse transcription primers is not confirmed, the plurality of stem-loop reverse transcription primers by a repeat process until the modified plurality of stem-loop reverse transcription primers demonstrates the multi-specificity and operatively quantifies the plurality of target miRNAs simultaneously.
4 more elements
Dependencies are inferred from the claim text.