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A kit, method and quality control method for simultaneous quantification of dna and rna

A quality control and quality technology, applied in the field of molecular biology, can solve the problems of increased background of detection results, decreased efficiency of NGS library construction, difficulty in RNA template library construction, etc.

Active Publication Date: 2021-01-29
HELITEC LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Nucleic acids in clinical samples are often stored at room temperature for a long time, and the nucleic acids, especially RNA, are degraded in large quantities, making RNA template library construction difficult
The commonly used formalin fixation process for clinical samples can cause cross-linking or damage to nucleic acids, which reduces the efficiency of NGS library construction and increases the background of test results.

Method used

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  • A kit, method and quality control method for simultaneous quantification of dna and rna
  • A kit, method and quality control method for simultaneous quantification of dna and rna
  • A kit, method and quality control method for simultaneous quantification of dna and rna

Examples

Experimental program
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Effect test

Embodiment 1

[0127] This example describes the kit of the present invention for the simultaneous quantification of DNA and RNA in a sample containing nucleic acid. Among them, the housekeeping gene selected is the human CHMP2A gene, the shorter intron in the CHMP2A gene is selected as intron No. 3 (the number of bases is 75), and the longer intron is intron No. 2 (the number of bases is 1608). The composition of the kit of the present invention is shown in Table 1, and the sequences of amplification primers and probes in the kit of the present invention are shown in Table 2. Wherein, the amplification primer 1 and the amplification primer 2 can anneal to the nucleic acid sequences of the two exons adjacent to the No. 3 intron in the CHMP2A gene.

[0128] Table 1 Composition of the kit.

[0129]

[0130] Table 2 Sequences of amplification primers and probes

[0131]

Embodiment 2

[0133] This example describes the method of using the kit described in Example 1 to simultaneously quantify DNA and RNA in a sample containing nucleic acid, that is, the method of using the kit described in Example 1. The equipment used in the method described in this embodiment is a fluorescent quantitative PCR instrument, and the specific steps are as follows:

[0134] Reagent pretreatment before the first use: Add 2 tubes of 50×Primer&Probe Mix to 2×PreQC DNA and 2×PreQC RNA respectively and mix well, labeled as 2×PreQC DNA Mix and 2×PreQC RNAMix.

[0135] 1) According to the following tables 3 and 4, prepare and mix the reagents:

[0136] Table 3 Quantitative PCR system without reverse transcription

[0137] components volume 2×PreQC DNA Mix 5.0 μL Nuclease-free deionized water 4μL Nucleic acid samples to be tested 1μL total capacity 10.0 μL

[0138] Table 4 Reverse transcription quantitative PCR system

[0139] comp...

Embodiment 3

[0145] This example describes the process of evaluating the quality of the nucleic acids of a batch of clinical tumor tissue samples: the samples are the total nucleic acids extracted from 244 tumor tissues, and the quantitative PCR values ​​are measured with the kit described in Example 1, and the DNA produced by ThermoFisher Qubit quantifies DNA and RNA. Nucleic acid is used for the single-end anchoring method targeting NGS library construction using RNA and DNA as templates, and its detection range includes a gene that is abundant and stable in various types of tissues as an internal reference. The amplicon of the gene begins at the marginal region of one exon, and the signal from the DNA template is measured with sequencing reads from the contiguous intron, and the signal from the RNA template is measured with sequencing reads from the exon located at the opposite end of the intron. Signal, the ratio of the two signals is used to measure the RNA detection quality of NGS li...

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Abstract

The invention relates to a kit, a method and a quality control method for simultaneously quantifying DNA and RNA in a sample containing nucleic acid, belonging to the field of molecular biology. In the kit of the present invention, the nucleic acid comprises a housekeeping gene, the housekeeping gene comprises a first intron and two exons adjacent to the first intron, and the two exons are respectively the first exon and the second exon Two exons; the kit comprises amplification primers capable of specifically annealing to the nucleic acid sequences of the first exon and the second exon, a first hybridization probe capable of hybridizing with the first intron, and a first hybridization probe capable of hybridizing with the second exon A second hybridization probe hybridizing to an exon or a second exon, with different labels on the first hybridization probe and the second hybridization probe. The present invention also compares the measured value of simultaneous quantification of DNA and RNA with the detection value of high-throughput sequencing for quality control, and provides a double quality control method for DNA and RNA quality, which is suitable for quickly and conveniently evaluating whether the sample quality is suitable for operation High-throughput sequencing genetic testing is required for high-throughput sequencing and high cost.

Description

technical field [0001] The invention relates to a kit, a method and a quality control method for simultaneously quantifying DNA and RNA, belonging to the field of molecular biology. Background technique [0002] Next-generation sequencing (NGS) technologies have revolutionized the field of genomics over the past decade. Each NGS run typically generates gigabytes of sequence information on hundreds of thousands to billions of DNA templates in parallel per sequencing run. The current cost for sequencing the human genome has reached the $1,000 benchmark. The low cost and high throughput of NGS technology have enabled the use of nucleic acid sequencing as a clinical tool. [0003] However, many challenges remain to achieve the desired cost, speed, analytical sensitivity, and accuracy required for clinical application of NGS. Clinical samples, such as biopsy samples and formalin-fixed paraffin-embedded (FFPE) samples, provide only a small amount of starting material. NGS libr...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/6851
CPCC12Q1/6851C12Q2531/113C12Q2545/101C12Q2563/107
Inventor 陈力郑宗立高彦秋何云蔚陈淼赖家嘉
Owner HELITEC LTD