# US 12,767,987 B2 · Method for screening multiplex reverse transcription primer combinations for quantifying multiple mirnas

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- Number: US 12,767,987 B2
- Document ID: US12767987B2
- Status: Granted
- Kind: B2
- Application number: 18/797,835
- Filed: 2024-08-08
- Published: 2024-11-28
- Granted: 2026-10-20
- Earliest claimed: 2023-05-06
- Assignees: Miracle Biotechnology Inc.
- Applicants: Miracle Biotechnology Inc.
- Inventors: Chang Su; Yuan Cao; Mengjing Cao; Demei Ma
- Web page: https://patentgrep.com/patent/US12767987B2

## CPC

- C12Q 1/6883: for diseases caused by alterations of genetic material
- C12N 15/1096: {cDNA Synthesis; Subtracted cDNA library construction, e.g. RT, RT-PCR}
- C12Q 1/6825: Nucleic acid detection involving sensors
- C12Q 1/6851: Quantitative amplification
- G01N 21/6486: {Measuring fluorescence of biological material, e.g. DNA, RNA, cells (G01N21/6428 takes precedence)}
- C12Q 2600/178: miRNA, siRNA or ncRNA
- C12Q: MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS (immunoassay G01N33/53); COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- G01N: INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES (measuring or testing apparatus or processes other than immunoassay, involving enzymes or microorganisms C12M, C12Q)

## 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.

## Claims

### Claim 1

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.

### Claim 2 (depends on 1)

The method according to claim 1, wherein step i) of the screening process comprises:
a hybridization reaction wherein each of the plurality of stem-loop reverse transcription primers is hybridized to at least one of the plurality of target miRNA to form a collection of at least one type of miRNA-stem-loop reverse transcription primer complex; and
an extension reaction wherein at least one type of cDNA is formed by extending a 3′ of the stem-loop reverse transcription primer in the collection of at least one type of miRNA-stem-loop reverse transcription primer complex.

### Claim 3 (depends on 2)

The method according to claim 2, wherein the step iii) of the screening process comprises:
adding a combination of forward primers and at least one reverse primer to each of the collections of reverse transcription products from step ii); wherein each of the forward primers has a sequence same to a 5′ sequence of one of the plurality of target miRNAs;
adding a combination of probes to each of the collections of reverse transcription products from step ii); wherein each of the probes has a sequence same as a portion of one of the target miRNA, and each of the probes has a fluorescent reporter group different from the fluorescent reporter groups of the other probes, and each of the probes has a quencher group suppressing a fluorescent light produced by the fluorescent reporter group;
separately amplifying each of the collections of reverse transcription products from step ii) with the combination of forward primers, the at least one reverse primer, and the combination of probes to separately form a collection of multiplex qPCR products for each of the collections of reverse transcription products;
detecting a strength of the fluorescent light produced by the fluorescent reporter groups of the combination of probes in each of the collections of multiplex qPCR products; and
quantifying each of the plurality of target miRNAs based on the strength of the fluorescent light detected in each of the collections of multiplex qPCR products.

### Claim 4 (depends on 3)

The method according to claim 3, wherein the quantification result of each of the plurality of target miRNA comprises a Ct value.

### Claim 5 (depends on 1)

The method according to claim 1, wherein the stem-loop sequences of at least two of the plurality of stem-loop reverse transcription primers selected in step a) are the same.

### Claim 6

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.

### Claim 7 (depends on 6)

The method according to claim 6, wherein 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 are modified by a repeat process comprising:
providing a modified plurality of stem-loop reverse transcription primers by repeating the 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 stem-loop reverse transcription primer is complimentary; and
repeating 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 each of the plurality of target miRNAs simultaneously.

### Claim 8 (depends on 6)

The method according to claim 6, wherein the step of performing the singleplex reverse transcription process comprises:
a hybridization reaction, wherein each of the plurality of stem-loop reverse transcription primers is hybridized to at least one of the plurality of target miRNAs to form a collection of at least one type of miRNA-stem-loop reverse transcription primer complex; and
an extension reaction, wherein at least one type of cDNA is formed by extending a 3′ of the stem-loop reverse transcription primer against the at least one of the plurality of target miRNAs in the at least one type of miRNA-stem-loop reverse transcription primer complex.

### Claim 9 (depends on 6)

The method according to claim 6, wherein the step of performing the multiplex qPCR amplification process comprises:
adding a combination of forward primers and at least one reverse primer to each of the collections of reverse transcription products; wherein each of the forward primers has a sequence same to a 5′ sequence of one of the plurality of target miRNAs;
adding a combination of probes to each of the collections of reverse transcription products; wherein each of the probes has a sequence same as a portion of one of the target miRNA, and each of the probes has a fluorescent reporter group different from the fluorescent reporter groups of the other probes, and each of the probes has a quencher group suppressing a fluorescent light produced by the fluorescent reporter group;
separately amplifying each of the collections of reverse transcription products with the combination of forward primers, the at least one reverse primer, and the combination of probes to separately form a collection of multiplex qPCR products for each of the collections of reverse transcription products;
detecting a strength of the fluorescent light produced by the fluorescent reporter groups of the combination of probes in each of the collections of multiplex qPCR products; and
quantifying each of the plurality of target miRNAs based on the strength of the fluorescent light detected in each of the collections of multiplex qPCR products.

### Claim 10 (depends on 7)

The method according to claim 7 further comprising a sensitivity test of the plurality of stem-loop reverse transcription primers, wherein the sensitivity test comprises:
1) preparing a plurality templates of mixed target miRNAs having a concentration gradient, wherein each of the plurality templates of mixed target miRNAs has a concentration different from the other templates of mixed target miRNAs;
2) separately performing a process of singleplex reverse transcription on each of the plurality templates of mixed target miRNAs with each of the plurality of stem-loop reverse transcription primers and collecting a collection of reverse transcription products from each of the processes of singleplex reverse transcription;
3) separately performing a process of multiplex qPCR using each of the collections of reverse transcription products from step 2), and obtaining a collection of quantification results of the plurality templates of mixed target miRNAs from the processes of multiplex qPCR;
4) arranging the collection of quantification results by each of the plurality of stem-loop reverse transcription primers; and
5) determining the lowest concentration of the mixed target miRNAs template for each of the plurality of stem-loop reverse transcription primers based on the arranged quantification results.

### Claim 11 (depends on 6)

The method according to claim 6, wherein the stem-loop sequence of at least two of the plurality of stem-loop reverse transcription primers are the same.

### Claim 12 (depends on 6)

The method according to claim 6, wherein the stem-loop sequence of all the plurality of stem-loop reverse transcription primers are the same.

### Claim 13 (depends on 6)

The method according to claim 6, wherein the plurality of target miRNAs comprises a combination of any two or more of hsa-miR-210-3p, hsa-miR-126-3p, hsa-miR-205-5p and hsa-miR-486-5p.

### Claim 14 (depends on 6)

The method according to claim 6, wherein the plurality of target miRNAs comprises a combination of any two or more of miR-16-5p, miR-34c-5p, miR-9-3p and miR-9-5p.

### Claim 15

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.

### Claim 16 (depends on 15)

The method according to claim 15, 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 such that the lengths of the anchor sequences of at least two of the plurality of stem-loop reverse transcription primers are different from each other.

### Claim 17 (depends on 16)

The method according to claim 16, wherein the screening step 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.