PCR analysis method for quantitative detection of microRNA
A quantitative detection and analysis method technology, applied in the field of detection and analysis, can solve problems such as increased cost and design complexity, and achieve the effects of low cost, reduced detection error, and wide application range
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Embodiment 1
[0052] The analysis method of let-7a (5′-UGAGGUAGUAGGUUGUAUAGUU-3′) is detected by one-step real-time quantitative PCR based on the principle of base stacking hybridization. The detection principle is as follows: figure 1 shown. TaqDNA polymerase and double-stranded DNA-specific dye SYBR Green I were used. The functional sequence on the DNA amplification template is the same sequence as the reverse primer (5′-GGCTAAGACAGATGCTC-3′), the target let-7a binding sequence (5′-AACTATACAACCTACTACCTCA-3′) and the forward primer binding sequence (5′ -TCGCCT-3').
[0053] The following specific implementation of let-7a detection will further illustrate the present invention. For the experimental methods that do not indicate the specific conditions, usually follow the conventional conditions or the conditions suggested by the manufacturer. The specific operation steps are as follows: Add 2 μL of reaction buffer solution (50 mM Tris-HCl, 20 mM potassium chloride, 10 mM ammonium sulfate,...
Embodiment 2
[0061] Using one-step real-time quantitative PCR based on the principle of base stacking hybridization to detect miR-141, miR-141 (5′-UAACACUGUCUGGUAAAGAUGG-3′) analysis method, the detection principle is as attached Figure 7 shown. TaqDNA polymerase and double-stranded DNA binding dye SYBR Green I were used. The functional sequence on the DNA amplification template is the same sequence as the reverse primer (5′-GGCTAAGACAGATGCTC-3′), the target miR-141 binding sequence (5′-CCATCTTTACCAGACAGTGTTA-3′) and the forward primer binding sequence (5′ -TCGCACT-3′).
[0062] The following specific implementation of miR-141 detection will further illustrate the present invention. For the experimental methods that do not indicate the specific conditions, usually follow the conventional conditions or the conditions suggested by the manufacturer. The specific operation steps are as follows: Add 2 μL of reaction buffer solution (50 mM Tris-HCl, 20 mM potassium chloride, 10 mM ammonium s...
Embodiment 3
[0065] An analysis method for detecting miR-21 (5′-UAGCUUAUCAGACUGAUGUUGA-3′) by one-step real-time quantitative PCR based on the principle of base stacking hybridization, the detection principle is as attached Figure 8 shown. TaqDNA polymerase and double-stranded DNA binding dye SYBR Green I were used. The functional sequence on the DNA amplification template is the same sequence as the reverse primer (5′-GGCTAAGACAGATGCTC-3′), the target miR-21 binding sequence (5′-TCAACATCAGTCTGATAAGCTA-3′) and the forward primer binding sequence (5′ -TCGCACT-3′).
[0066] The following specific implementation of miR-21 detection will further illustrate the present invention. For the experimental methods that do not indicate the specific conditions, usually follow the conventional conditions or the conditions suggested by the manufacturer. The specific operation steps are as follows: Add 2 μL of reaction buffer solution (50 mM Tris-HCl, 20 mM potassium chloride, 10 mM ammonium sulfate, ...
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