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Tomato total RNA extraction method suitable for microRNAs analysis

An extraction method and tomato technology are applied in the field of extracting total RNA from tomato tissue, which can solve the problems of expensive economic pressure of kits, large amount of reagents and experimental materials, and difficulty in popularization and application, and achieve the effect of saving experimental costs.

Inactive Publication Date: 2012-08-22
CHONGQING UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

After several years of development, although some tomato total RNA extraction techniques have been formed, such as the guanidine isothiocyanate method, these traditional methods generally require a large amount of reagents and experimental materials, and the cost is high, and the operation is slightly careless. RNA degradation
Especially for experiments with less demand for RNA, it often results in unnecessary waste of experimental materials and reagents
Although RNA extraction kits have excellent extraction effects, such as the RNeasy Plant Mini Kit from QIAGENE, the high price of the kits often brings great economic pressure to the research work, and it is difficult to be widely used

Method used

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  • Tomato total RNA extraction method suitable for microRNAs analysis
  • Tomato total RNA extraction method suitable for microRNAs analysis
  • Tomato total RNA extraction method suitable for microRNAs analysis

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0019] Example 1: Add 500 μL of phenol and 500 μL of TLES buffer into a 5 mL centrifuge tube, and fully preheat at 80° C. 0.07 g of tomato leaf material ground into powder in liquid nitrogen was transferred to the above-mentioned tube, kept at 80° C. for 30 s, and vigorously vortexed for 1 min. Then add 1 / 2 volume of chloroform / isoamyl alcohol, vortex and mix thoroughly, and then centrifuge at 12,000g for 5min at 4°C. Transfer the supernatant to a new centrifuge tube and extract once with an equal volume of chloroform / isoamyl alcohol. Transfer the supernatant to a new centrifuge tube, add an equal volume of isopropanol, precipitate at room temperature for 30 minutes, and centrifuge at 12,000 g for 10 minutes at 4°C. Remove the supernatant, dissolve the precipitate in an appropriate amount of DEPC water, add 1 / 10 volume of 3mol / L sodium acetate and 2 volumes of absolute ethanol. After precipitation at -80°C for 1 hour or overnight at -20°C, centrifuge at 12,000 g for 30 minut...

Embodiment 2

[0020] Example 2: A larger amount of total RNA from plant materials is extracted by appropriately increasing the amount of reagent used. Add 5mL phenol and 5mL TLES buffer into a 50mL centrifuge tube to extract 0.5g tomato leaf material, and the operation steps are the same as in Example 1.

[0021] The effect of the total RNA extracted in the above examples is shown in Table 1 and attached to the instructions. figure 1 , 2 .

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Abstract

The invention provides a tomato total RNA extraction method suitable for microRNAs analysis, which is characterized in that by the step of extracting total RNA through 80-DEG C preheated phenol and a TLES buffer solution (100mM tris(hydroxymethyl)aminomethane (Tris pH 8.0), 10mM lithium chloride, 10mM ethylene diamine tetraacetic acid (EDTA pH 8.0), 1 percent sodium dodecyl sulfonate (SDS, w / v)) by combining isopropyl alcohol and sodium acetate / alcohol, large-segment RNA can be separated, and micromolecular RNA can be effectively settled. The extracted RNA has high quality and good integrity, and can be used for the gene separation and expression analysis of RT-PCR, northern hybrid and the like, and the research of micromolecular RNA of microRNAs and the like. The method has the advantages of simpleness, low cost, high efficiency and the like and is easy to operate and suitable for being used in a molecular biology experiment.

Description

1. Technical field [0001] The invention relates to a method for extracting plant total RNA, in particular to a method for extracting total RNA from tomato tissue. 2. Background technology [0002] RNase contamination is one of the main reasons for the difficulty in RNA extraction, and RNase usually comes from two sources, exogenous RNase and endogenous RNase. The former comes from the utensils, reagents and solutions of the RNA preparation process, and the researchers themselves, etc., and can reduce the impact of exogenous RNases by means such as preparing reagents and handling utensils with DEPC water, keeping the environment clean and operators changing gloves frequently . The endogenous RNase is carried by the plant material itself, which can only be overcome by improving the extraction method itself. Due to the ubiquity and high stability of RNase, it often brings great difficulties to the isolation of high-quality RNA. Therefore, establishing a good RNA extraction t...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/10
Inventor 杨迎伍李正国吴玉邓伟
Owner CHONGQING UNIV