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Internal reference gene, primer pair and application of purple cabbage

An internal reference gene, purple cabbage technology, applied in the field of molecular biology, can solve the problems that hinder the study of gene expression characteristics and gene function analysis, the difficulty of realizing gene expression level correction and standardization, and the inability to express level research and comparison, etc., to achieve shortening The effect of detection time, improved confidence, improved stability and reliability

Active Publication Date: 2021-04-06
INST OF AGRO FOOD SCI & TECH CHINESE ACADEMY OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The lack of internal reference genes makes it difficult to correct and standardize gene expression levels, and it is also impossible to study and compare the expression levels of genes under different conditions and developmental stages, which greatly hinders the research on the expression characteristics and gene functions of various genes in purple cabbage. Analysis

Method used

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  • Internal reference gene, primer pair and application of purple cabbage
  • Internal reference gene, primer pair and application of purple cabbage
  • Internal reference gene, primer pair and application of purple cabbage

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0043] Cloning of embodiment 1 purple cabbage 18SrRNA gene

[0044] 1.1 Extraction of total RNA from purple cabbage:

[0045]Total RNA was extracted using a plant RNA extraction kit (RNAprep Pure Plant Kit, DP441 TIANGEN). Immediately after taking fresh materials, wrap them in tin foil and store them in liquid nitrogen, and store them in a -80°C refrigerator for later use. The pipette tips, centrifuge tubes and water used in the extraction process were treated with DEPC overnight and autoclaved at 121°C for 40 minutes for later use. The mortar and pestle used for grinding samples were sealed with tinfoil in advance and autoclaved at 121 °C for 40 min. The specific method is as follows: Take about 100 mg of fresh plant samples and grind them fully in liquid nitrogen, add the powder into a 1.5 mL EP tube pre-cooled in liquid nitrogen, and quickly add 500 μL of lysate SL (before use, add β- mercaptoethanol), immediately vortexed vigorously to mix, and allowed to stand at room ...

Embodiment 2

[0056] Example 2. Real-time fluorescent quantitative PCR design and fluorescent quantitative PCR detection

[0057] Based on the nucleotide sequence of the internal reference gene obtained in Example 1, special fluorescent quantitative primers were designed according to the primer design principles of real-time fluorescent quantitative PCR, and the amplified fragment was 85 bp. The pair of fluorescent quantitative specific primers are the real-time fluorescent quantitative PCR primers (as shown in SEQ ID NO: 4 and 5): the forward primer is: q18SrRNA-F (SEQ ID NO: 4): 5'-TTAGGTTTCCGTGCAGGAGT- 3', the reverse primer is: q18SrRNA-R (SEQ ID NO: 5): 5'-CATGCGATGACTTGTCCG-3'. Samples were taken in triplicate as independent experimental replicates. The cDNA templates were diluted 50-fold for the following fluorescent quantitative PCR reactions.

[0058] Use Takara FAST qPCR Kit kit (RR430B Takara) was used for fluorescent quantitative PCR. Firstly, the reverse transcription prod...

Embodiment 3

[0063] Example 3 Purple cabbage 18SrRNA gene expression stability analysis

[0064] According to the RNA extraction method of 1.1 in the above-mentioned embodiment 1, the total amount of different purple cabbage samples (sample numbers 1, 2, 3, 4, 5, 6) and samples of different developmental stages of purple cabbage (P1, P2, P3 and P4) were extracted. RNA, and synthesize the first strand of cDNA according to the method of 1.2 in Example 1. Three replicates of material were taken for each sample as independent experimental replicates. The cDNA templates were diluted 50-fold for the following fluorescent quantitative PCR reactions. The system and procedure of the fluorescent quantitative PCR reaction were carried out as described in Example 2. Amplification curve such as Figure 4 As shown, the expression of 18SrRNA gene is very stable among different samples, and the Ct values ​​are 20.33±0.13, 20.18±0.02, 19.86±0.13, 19.91±0.05, 20.80±0.07, 20.03±0.14, 19.96±0.32, 19.92±0.0...

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Abstract

The invention discloses an internal reference gene of purple cabbage, the base sequence of which is shown in SEQ ID NO:1. The invention discloses a purple cabbage internal reference gene amplification primer pair. The amplification primer pair is the base sequence shown in SEQ ID NO: 2 and 3. The invention discloses a pair of real-time fluorescent quantitative primers for internal reference genes of purple cabbage. The real-time fluorescent quantitative primer pair is: the base sequences shown in SEQ ID NO: 4 and 5. The invention discloses a kit for detecting the relative expression level of functional genes of purple cabbage, which comprises a pair of real-time fluorescent quantitative primers. The invention discloses the use of an internal reference gene or a real-time fluorescent quantitative primer for the detection of the relative expression levels of purple cabbage functional genes in different development stages of purple cabbage and in different varieties of purple cabbage. The invention solves the current situation that there is no internal reference gene in the existing quantitative PCR detection of purple cabbage, and improves the stability and reliability of gene expression analysis in detection of purple cabbage by using real-time fluorescent quantitative PCR technology.

Description

technical field [0001] The present invention belongs to the technical field of molecular biology, and relates to internal reference gene, primer pair and application of purple cabbage, in particular to the sequence and cloning method of internal reference gene 18SrRNA of purple cabbage and its amplification primer as internal reference gene and its application in RT-qPCR . Background technique [0002] Purple cabbage (Brassica oleracea L.var.capitata f.rubra DC.) is a variety of Brassica oleracea in the family Brassicaceae. Purple cabbage is rich in nutrition, especially rich in vitamin C, more vitamin E, vitamin B, and rich in anthocyanins, minerals and cellulose, which are very popular among people. The main nutritional components of purple cabbage are similar to those of head cabbage, but they contain higher vitamins and minerals than head cabbage, and contain a lot of natural pigments. Therefore, it is recognized that the nutritional value of purple cabbage is higher th...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/6895C12N15/11
CPCC12Q1/6895C12Q2600/158C12Q2600/166C12Q2600/178
Inventor 王志东吴杰张洁王丽金孙倩倩
Owner INST OF AGRO FOOD SCI & TECH CHINESE ACADEMY OF AGRI SCI
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