Method for extracting total RNA (Ribonucleic Acid) from rose plant tissues

A technology of plant tissue and Rosa, which is applied in the field of extracting high-quality total RNA, can solve the problems of unsatisfactory, low broad-spectrum, poor integrity of total RNA, etc., and achieve the effect of improving quality and yield

Pending Publication Date: 2022-07-22
JINGCHU UNIV OF TECH
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  • Abstract
  • Description
  • Claims
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Problems solved by technology

Through multiple experiments, the applicant found that these methods are not thorough in removing secondary metabolites in samples, and the quality and efficiency of RNA extraction are low, the stability is poor, and the broad spectrum is small, which cannot meet the requirements of RT-PCR, RT-qPCR, Northern hybridization, The needs of molecular biology experiments such as cDNA library construction and transcriptomic analysis, while the applicant consulted the literature, did not see the subsequent extensive and large-scale application of these methods
In addition, although the total RNA extraction kits from polysaccharide and polyphenol plant tissues sold in the market today can extract high-quality total RNA from Rosa plant tissues (Liu Hangcheng et al., 2021), the integrity of the total RNA is not good, so it can be obtained The efficiency is low and the cost is high, only suitable for the extraction of a small amount of RNA

Method used

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  • Method for extracting total RNA (Ribonucleic Acid) from rose plant tissues
  • Method for extracting total RNA (Ribonucleic Acid) from rose plant tissues
  • Method for extracting total RNA (Ribonucleic Acid) from rose plant tissues

Examples

Experimental program
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Effect test

Embodiment 1

[0062] Embodiment 1, the method that extracts total RNA from Rosa plant tissue

[0063] In this example, total RNA was extracted from the tissues (roots, stems, leaves, flowers and fruits) of wild rose, Chinese rose ('Yueyuefen' variety), rose ('Zizhi' variety), and various tissues (roots, stems, leaves, flowers and fruits), including The following steps:

[0064] (1) Weigh 0.2g of roots, stems, leaves, flowers and fruits, respectively, into a mortar pre-cooled by liquid nitrogen, add 0.01g of PVPP (crospovidone, cross-linked polyvinylpyrrolidone), and place in liquid nitrogen fully ground into fine powder;

[0065] (2) Quickly transfer the sample powder (fine powder) to a 1.5 mL RNase-free centrifuge tube (RNase-free centrifuge tube) containing 0.6 mL of solution A, vortex to mix, let stand for 3 minutes, and centrifuge at 12,000 rpm for 5 minutes;

[0066] (3) Aspirate the supernatant into a new 1.5mL RNase-free centrifuge tube, add an equal volume of solution B, shake and...

Embodiment 2

[0091] Example 2: Detection of cDNA quality by reverse transcription synthesis of total RNA from different tissues of Rhododendron chinensis

[0092] 1. Take the total RNA of the roots, stems, leaves, flowers and fruits of Rosa japonica obtained in Example 1 as a template, and utilize Novizan Biotechnology Co., Ltd. II Reverse Transcriptase Reverse Transcription Kit performs reverse transcription (RT) to synthesize the root cDNA, stem cDNA, leaf cDNA, flower cDNA and fruit cDNA respectively, and store at -20°C for later use.

[0093] 2. Detect and analyze the cDNA of each tissue of Rose thorn in step 1 by 1% agarose gel electrophoresis. Electrophoresis imaging results ( figure 2 ) shows that the cDNA bands are mainly distributed between 0.5 and 1.0 kb, with a wide distribution range and high concentration, indicating that the cDNA synthesized by reverse transcription of total RNA from different tissues of Rhizoma thorne extracted by the method of the present invention is of...

Embodiment 3

[0094] Example 3: Amplification of Rose RcACTIN Gene Fragment by RT-PCR

[0095] 1. The total RNA of Chinese rose root, stem, leaf, flower and fruit obtained in Example 1 is used as a template, using Novozyme Biotechnology Co., Ltd. II Reverse Transcriptase Reverse Transcription Kit performs reverse transcription (RT) to synthesize cDNA of rose root, stem, leaf, flower and fruit respectively, and store at -20°C for future use.

[0096] 2. Using the cDNA of each rose tissue obtained in step 1 as a template, RcACTIN-F (5'-TGGGACTGGAATGGTCAA-3') and RcACTIN-R (5'-GATGCTAAGATAGAGCCTCCG-3') as primers to carry out polymerase chain reaction ( PCR) to amplify the fragment of the rose RcACTIN gene (RcHm_v2.0_Chr3g0466761).

[0097] PCR reaction system: 25 μL 2×Taq PCR master mix, 5 μL cDNA template, 10 μL 1 μM F primer and 10 μL 1 μM R primer.

[0098] PCR reaction program: pre-denaturation at 94°C for 5min, denaturation at 94°C for 30sec, annealing at 58°C for 30sec, extension at ...

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Abstract

The invention discloses a method for extracting total RNA (Ribonucleic Acid) from rose plant tissues. In particular discloses a method for extracting total RNA (Ribonucleic Acid) from a rose plant tissue, the method comprises the following steps: grinding the rose plant tissue by liquid nitrogen, and performing continuous three times of splitting decomposition and extraction, the three times of splitting decomposition and extraction respectively adopt a solution A, a solution B and a solution C for splitting decomposition, and the components of the solution A, the solution B and the solution C are disclosed. The method provided by the invention effectively solves the problem of interference of secondary metabolites, proteins, DNA and the like on RNA extraction, and obviously improves the extraction quality and efficiency of total RNA of rose plant tissues. The method disclosed by the invention has the advantages of high efficiency, high quality, low cost, short period, good stability, simplicity in operation, easiness in wide application and the like, and the extracted total RNA is good in integrity, high in purity and high in yield and can be directly applied to downstream molecular biology experiments such as RT-PCR (Reverse Transcription-Polymerase Chain Reaction) and cDNA (Complementary Deoxyribonucleic Acid) library construction.

Description

technical field [0001] The invention belongs to the technical field of plant molecular biology, and in particular relates to a method for extracting high-quality total RNA from tissues such as roots, stems, leaves, flowers, fruits and the like of Rosa plants. Background technique [0002] The extraction of total RNA from plant tissue is the experimental basis for molecular biology researches such as gene cloning, expression, function and regulation. High-quality total RNA with high purity and integrity is the key to molecular biology experiments such as RT-PCR, RT-qPCR, Northern hybridization, cDNA library construction and transcriptomic analysis. RNA is a class of easily degradable molecules. To obtain complete RNA, the degradation of RNA by endogenous and exogenous ribonucleases (RNases) must be suppressed to the maximum extent during the extraction process. Compared with animals and microorganisms, it is more difficult than animals and microorganisms. For example, the ox...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/10C40B40/06
CPCC12N15/1003C40B40/06C12Q2527/125
Inventor 田雪军韦如健陈士李仔叶吴晶晶李昊璞徐佩琪徐艳熊兴鹏
Owner JINGCHU UNIV OF TECH
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