Reagent for high-quality extraction of RNA in mature corn kernels and embryos and use method thereof

Abstract

The invention relates to a reagent for high-quality extraction of RNA (Ribonucleic Acid) in mature corn kernels and embryos and an extraction method of the reagent. The reagent is named as RNA extraction auxiliary liquid, starting from the characteristic that corn kernels and embryos are rich in starch, protein, grease, polysaccharide, polyphenol and the like, the reagent for removing impurities in the mature corn kernels and embryos in the RNA extraction process and the extraction method are designed and researched and developed, in the initial stage of extraction, the impurities are removed as much as possible, smooth extraction in the subsequent process is guaranteed, and the extraction efficiency is improved. And finally, high-quality RNA is obtained. The method is suitable for RNA extraction in corn seeds and embryos, and the obtained high-quality RNA can be applied to subsequent PCR (Polymerase Chain Reaction), high-throughput sequencing and other experiments.

Description

Technical Field

[0001] The invention relates to the technical field of RNA extraction from corn kernels and embryos, and in particular to a reagent capable of extracting RNA from mature corn kernels and embryos with high quality, and a method for extracting RNA from mature corn kernels and embryos with high quality. Background Art

[0002] Corn is the largest crop in China in terms of planting area, integrating grain, cash crops and feed. With the continuous improvement of corn genome information, many genes regulating important agronomic traits of corn have been cloned, and the molecular breeding level of corn has been further improved. According to experimental requirements, when extracting some special materials that cannot germinate, or when taking corn embryos as the research object, only kernels or embryos can be used for extraction. However, corn kernels, especially corn embryos, contain a large amount of oil, protein, polysaccharides, polyphenols and other substances. When the conventional TRIzol method is used to extract RNA, a large amount of non-nucleic acid white precipitation is accompanied by the step of using isopropanol to precipitate RNA, which seriously reduces the quality of extracted RNA and makes it impossible to conduct subsequent experiments. With the increase of seed maturity, the content of oil, protein, polysaccharide and polyphenol substances accumulates, which has a greater impact on the extraction of high-quality nucleic acids from kernels and embryos. Therefore, this study invented a reagent for removing impurities in kernels and embryos during RNA extraction from mature corn kernels and embryos, as well as corresponding experimental methods, to solve the problem of extracting high-quality RNA from mature corn kernels and embryos, and provide a solid foundation for subsequent molecular experiments such as PCR and high-throughput sequencing. Summary of the invention

[0003] In view of the lack of high-quality reagents for extracting RNA from mature corn kernels and embryos in the prior art, the primary purpose of the present invention is to provide a reagent formula and corresponding use method for assisting the TRIzol method to extract high-quality RNA from mature corn kernels and embryos. Through this reagent and method, high-quality RNA can be effectively extracted from corn kernels or embryos to meet scientific research needs. BRIEF DESCRIPTION OF THE DRAWINGS

[0004] Figure 1 : Photos of RNA extracted from corn kernels and embryos using the conventional TRIzol method, i.e., without the RNA extraction auxiliary solution of this study, and dissolved in RNase-free DEPC-treated water; and photos of RNA extracted using the RNA extraction auxiliary solution of this study and dissolved in RNase-free DEPC-treated water. Also, agarose gel electrophoresis of RNA extracted with and without the RNA extraction auxiliary solution of this study at the same loading amount. DETAILED DESCRIPTION

[0005] Unless otherwise specified, the experimental materials and reagents used in the following implementations can be obtained from commercial sources. The mature corn kernels and embryos are the kernels and embryos of corn inbred line B73 after self-pollination for 35 days.

[0006] Maize inbred line B73 was collected and preserved for this experiment and cultivated and managed according to general field requirements.

[0007] The formula of nucleic acid extraction solution is as follows: Component A: PVP-40 3g; EDTA 3.6g; dissolved in 100mL Tris-HCl 8.0, add 1mL mercaptoethanol before use. Component B: Dissolve 10g SDS in distilled water and dilute to 100mL.

[0008] Example 1. Extraction of high-quality RNA from mature corn kernels or embryos

[0009] Mature corn seeds or embryos were completely ground into fine powder using liquid nitrogen, placed in a 2.0 mL centrifuge tube, and temporarily stored in liquid nitrogen.

[0010] Add 600 μL of component A to every 100 mg of seeds or embryos. Mix well and place on ice for 6 minutes, shaking every 2 minutes to mix well.

[0011] Add 40 μL of component B and place on ice for 4 minutes, shaking to mix every 2 minutes.

[0012] The mixture was centrifuged at 12,000 rpm in a refrigerated centrifuge at 4°C for 6 minutes, and the supernatant was aspirated.

[0013] Add 600 μL TRIzol, mix well, and place on ice for 8 minutes.

[0014] Centrifuge at 4°C, 10,000 rpm for 6 min and aspirate the supernatant.

[0015] Add an appropriate amount of DNase and leave at room temperature for 10 minutes.

[0016] Add 600 μL of chloroform, mix well, and place on ice for 5 minutes.

[0017] Centrifuge at 4°C, 10,000 rpm for 6 minutes and aspirate the supernatant.

[0018] Add 500 μL of isopropanol stored on ice and store at -20° for 15 minutes.

[0019] Centrifuge at 4°C, 10,000 rpm for 6 minutes and discard the waste liquid.

[0020] Add 1 mL of 70% ethanol, shake gently, centrifuge at 4°C, 10,000 rpm for 6 minutes, and discard the waste liquid.

[0021] Use a pipette to carefully remove the ethanol at the bottom of the centrifuge tube, and blow dry on a clean bench to remove residual ethanol.

[0022] Add RNase-free DEPC-treated water and dissolve at room temperature for 5 minutes. Use NanoDrop micro-spectrophotometer to detect RNA quality. The test results are shown in Table 1.

[0023] Use 1% agarose gel, 140V electrophoresis for 20 minutes to obtain the electrophoresis gel map of DNA quality detection, see Figure 1 .

[0024] Table 2. RNA quality measured using the NanoDrop micro-volume spectrophotometer

[0025]

[0026] As can be seen from Table 1, with grains as experimental objects, the RNA concentration extracted using the patented method was 168.5-228.1 ng / μL, the total amount was 16.85-22.81 μg, the A260 / A280 value was 1.98-2.04, and the A260 / A230 value was 1.57-1.63. However, using the conventional TRIzol method, the A260 / A230 value of the extracted grain RNA was only 0.25- 0.37; Taking embryos as experimental objects, the RNA concentration extracted by the patented method is 558.2-679.8ng / μL, the total amount is 55.82-67.98μg, the A260 / A280 value is 2.03-2.07, and the A260 / A230 value is 1.91-2.13. However, using the conventional TRIzol method, the A260 / A230 value of the embryo RNA extracted is only 0.24-0.32. This shows that compared with the conventional TRIzol method, the RNA extracted by the patented method is of high purity and good quality.

[0027] Depend on Figure 1 It can be seen that when the conventional TRIzol method is used to extract RNA from grains and embryos, insoluble white impurities are precipitated at the bottom of the centrifuge tube after the experiment is completed, while the DNA extracted using the patented method is completely dissolved in RNase-free DEPC-treated water ( Figure 1 -A, B). Using 1% agarose gel, adding the same amount of sample, and electrophoresing the extracted RNA at 140V for 20 minutes, it can be seen that the RNA extracted using the conventional method is of very poor quality and contains only one band. The RNA extracted using the patented method is of better quality, and the three RNA bands (28S, 18S, and 5S) are clearly distinguished ( Figure 1 -C,D).

[0028] Based on the above research results, it can be seen that the present invention is a reagent that can extract high-quality RNA from mature corn kernels and embryos, and a method that can extract high-quality RNA from mature corn kernels and embryos. The present invention provides a low-cost and highly operable solution for high-quality extraction of RNA from mature corn kernels and embryos, and will play an important role in the development research of corn kernels and embryos.

Claims

1. A reagent for extracting RNA from mature corn kernels and embryos with high quality and a method for using the same, characterized in that The formula of the nucleic acid extraction auxiliary liquid of this method and the corresponding usage method are as follows.

2. The RNA extraction auxiliary solution according to claim 1 is formulated as follows: Component A: 5 g PVP-40 (polyvinyl pyrrolidone); 3.6 g EDTA (ethylenediaminetetraacetic acid); dissolved in 100 mL Tris-HCl 8.0 (tris(hydroxymethylaminomethane) hydrochloride, pH=8.0), and 0.5 mL β-mercaptoethanol was added before use; Component B: 10 g SDS (sodium dodecyl sulfate) was dissolved in distilled water and the volume was adjusted to 100 mL.

3. The RNA auxiliary liquid formula according to claim 2 is used as follows when extracting RNA from mature corn seeds or embryos: freshly collected mature corn seeds or embryos are completely ground into fine powder using liquid nitrogen; 600 μL of component A is added to every 100 mg of seeds or embryos; after mixing, the mixture is placed on ice for 6 minutes and shaken every 2 minutes; 40 μL of component B is added, the mixture is placed on ice for 4 minutes and shaken every 2 minutes; the mixture is centrifuged in a refrigerated centrifuge at 4°C and 12000 rpm for 6 minutes, and the supernatant is aspirated; and subsequent operations are consistent with conventional extraction methods.

Images