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Method for fast detecting genome DNA residues in total RNA sample

A sample and genome technology, applied in the biological field, can solve the problems of genomic DNA contamination, whether the DNA has been removed completely cannot be known, and the quality of cDNA cannot be predicted.

Active Publication Date: 2018-11-06
INST OF VEGETABLE & FLOWERS CHINESE ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although most RNA extraction kits include a DNA removal step, it is not known whether the DNA is removed completely
In addition, in order to avoid residual genomic DNA contamination in RNA samples, some first-strand cDNA synthesis kits also add genomic DNA removal steps, but the quality of cDNA is unpredictable
Most importantly, there is no relevant literature report on the current method for the detection of genomic DNA residues in lily total RNA

Method used

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  • Method for fast detecting genome DNA residues in total RNA sample
  • Method for fast detecting genome DNA residues in total RNA sample
  • Method for fast detecting genome DNA residues in total RNA sample

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0052] Example 1 Genomic DNA Residual Detection in Total RNA Samples Extracted by Different Kits

[0053] Including the following steps:

[0054] (1) Total RNA sample extraction

[0055] Three kinds of RNA extraction kits produced by different companies were selected, and the total RNA was extracted from the leaves of 6 plants according to the instructions of the respective kits. The extracted total RNA samples were detected by 1.2% agarose gel electrophoresis and QuawellQ3000 detection, the results are shown in Figure 4 ,From Figure 4 It can be seen that the extracted 18 total RNA samples have good integrity and no protein contamination. Moreover, it is impossible to judge with the naked eye whether there is genomic DNA contamination in the RNA sample only from the figure.

[0056] (2) Detection of genomic DNA residues in RNA samples

[0057] Use genomic DNA residual detection primers (LDRG-F / LDRG-R) to perform PCR amplification on the extracted RNA, PCR reaction syste...

Embodiment 2

[0060] Example 2 Genomic DNA residue detection in cDNA samples

[0061] Including the following steps:

[0062] (1) Total RNA sample extraction

[0063] With embodiment 1.

[0064] (2) First strand cDNA synthesis

[0065] Using two first-strand cDNA synthesis kits from different companies (labeled as I and II respectively; I does not contain genomic DNA removal steps, and II contains genomic DNA removal steps), respectively refer to the instructions of the respective kits, and analyze 18 total RNAs. Samples undergo first-strand cDNA synthesis.

[0066] (3) Detection of genomic DNA residues in cDNA samples

[0067] The obtained cDNA was used as a template, and the genomic DNA residue detection primers (LDRG-F / LDRG-R) were used for PCR amplification respectively. The PCR reaction system was 20 μL: template cDNA (about 1 ng / μL) 2 μL, LDRG-F Primer 0.5 μL, LDRG-RPrimer 0.5 μL, 2×Taq PCR Magic Mix 10 μL, ddH 2 O 7 μL.

[0068] The PCR reaction procedure is the same as in Exa...

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Abstract

The invention provides a method for fast detecting genome DNA residues in a total RNA sample. The method comprises the steps that 1, according to genetic information of a to-be-detected biological material, a gene containing one or more intron sequence is selected as a target, and for the intron sequence, PCR primers are designed; 2, the total RNA of the to-be-detected biological material is extracted, the total RNA or cDNA obtained through reverse transcription of the total RNA is used as a template, and the primers in the step 1 are used for PCR amplification; 3, a PCR amplification productis analyzed. According to the method, only one pair of the primers are designed, so that whether or not the genome DNA residues exist in the total RNA and cDNA samples of the biological material can be detected simply, conveniently and fast; the method provides support for the accuracy of gene expression analysis and has important scientific significance for promoting fundamental research of the molecular biology.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a method for rapidly detecting genomic DNA residues in total RNA samples. Background technique [0002] Lily (Lilium spp.) is a multi-purpose plant with a long history of cultivation in my country for medicine, food and appreciation, and one of the top five fresh cut flowers in the world. With the continuous development of modern molecular biology, lily color (Yamagishi M, Yoshida Y, NakayamaM. The transcription factor LhMYB12 determines anthocyanin pigmentation in thetepals of Asiatic hybrid lilies (Lilium spp.) and regulates pigmentquantity. Molecular Breeding, 2012, 30 (2 ):913-925.), Huaxiang (Du F, Fan JM, Wang T, etal. Identification of differentially expressed genes in flower, leaf and bulbscale of Lilium oriental hybrid 'Sorbonne' and putative control network forscent genes. BMC Genomics, 2017 , 18(1):899.), development (Yang PP, Xu LF, Xu H, etal. Histological and transcrip...

Claims

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Application Information

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IPC IPC(8): C12Q1/6895C12Q1/686C12N15/11
CPCC12Q1/686C12Q1/6895
Inventor 袁素霞刘春
Owner INST OF VEGETABLE & FLOWERS CHINESE ACAD OF AGRI SCI
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