CN115247204A - Method for detecting lncRNA subcellular localization based on qRT-PCR technology - Google Patents

Method for detecting lncRNA subcellular localization based on qRT-PCR technology Download PDF

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CN115247204A
CN115247204A CN202210137311.7A CN202210137311A CN115247204A CN 115247204 A CN115247204 A CN 115247204A CN 202210137311 A CN202210137311 A CN 202210137311A CN 115247204 A CN115247204 A CN 115247204A
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蒋朋飞
朱华
张玉阳
张丽芳
陈平东
崔洲莹
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Abstract

The invention discloses a method for detecting the subcellular localization of lncRNA based on a qRT-PCR technology, and relates to the technical field of biological detection; the method comprises the following steps: (1) nuclear and cytoplasmic separation: cracking the cell to be detected by using a cell membrane lysate containing NP-40 to obtain a nucleus part and a cytoplasm part; (2) RNA extraction: respectively extracting total RNA, nuclear RNA and cytoplasmic RNA from cells which are not separated from nucleoplasm and the nuclear part and the cytoplasmic part by a Trizol extraction method; (3) cDNA Synthesis: reverse transcribing said nuclear RNA, said cytoplasmic RNA and said total RNA to synthesize nuclear cDNA, cytoplasmic cDNA and total cDNA, respectively; (4) qRT-PCR detection; and (5) identifying the lncRNA location. According to the invention, GAPDH is used as a qualitative internal reference, U6 is used as a nuclear internal reference, and the lncRNA subcellular localization is successfully detected by utilizing qRT-PCR technology.

Description

Method for detecting lncRNA subcellular localization based on qRT-PCR technology
Technical Field
The invention relates to the technical field of biological detection, in particular to a method for detecting lncRNA subcellular localization based on qRT-PCR technology.
Background
Long non-coding RNA (lncRNA) is RNA with the length of more than 200 nucleotides, lacks or has no open reading coding frame, and can play an important role in the occurrence and development process of a plurality of human cancers as a cancer suppressor or cancer promoter. The specific way incrna functions includes multiple levels of chromatin modification, transcription, and post-transcriptional regulation. However, the regulation mode of lncRNA is closely related to the distribution of lncRNA in subcellular. For example, lncRNA localized in the nucleus may affect the expression of target genes by regulating histone modification and chromatin remodeling, recruit transcription factors or inhibitors to the promoters of target genes to regulate transcription or bind with transcription-related proteins, and prevent the binding with target DNA; while lncRNA localized in the cytoplasm more affects the post-transcriptional biological processes.
Therefore, the detection of the subcellular localization of the lncRNA helps to further research the specific mechanism of the lncRNA, so as to be convenient for exploring the pathogenesis of the cancer from the angle of the lncRNA, thereby playing a positive role in the diagnosis and treatment of the cancer.
Disclosure of Invention
The invention aims to provide a method for detecting the location of lncRNA subcells based on a qRT-PCR technology, which aims to solve the problems in the prior art.
In order to achieve the purpose, the invention provides the following scheme:
the invention provides a method for detecting lncRNA subcellular localization based on qRT-PCR technology, which comprises the following steps:
(1) Separating nucleoplasm: cracking the cell to be detected by using a cell membrane lysate containing NP-40 to obtain a nucleus part, a cytoplasm part and a cell without nucleoplasm separation;
(2) RNA extraction: extracting total RNA, nuclear RNA and cytoplasmic RNA from the cells without nucleoplasm separation and the nuclear part and the cytoplasmic part respectively by using a Trizol extraction method;
(3) And (3) cDNA synthesis: reverse transcribing said nuclear RNA, said cytoplasmic RNA and said total RNA to synthesize nuclear cDNA, cytoplasmic cDNA and total cDNA, respectively;
(4) qRT-PCR detection: performing qRT-PCR detection by using GAPDH as a quality reference and U6 as a nuclear reference, and detecting lncRNA, GAPDH and U6 by using the nuclear cDNA, the quality cDNA and the total cDNA respectively to obtain 9 groups of Ct value data, namely total lncRNA, total GAPDH, total U6, nuclear lncRNA, nuclear GAPDH, nuclear U6, quality lncRNA, quality GAPDH and quality U6;
(5) Identification of lncRNA localization:
calculating four groups of data (a quality lncRNACt value-a quality GAPDH Ct value), (a total lncRNACt value-a total GAPDH Ct value), (a core lncRNACt value-a core U6 Ct value), and (a total lncRNACt value-a total U6 Ct value) as delta Ct;
calculating two groups of data (a quality lncRNACt value-a quality GAPDH Ct value) - (a total lncRNACt value-a total GAPDH Ct value), (a core lncRNACt value-a core U6 Ct value) - (a total lncRNACt value-a total U6 Ct value) as Δ Ct;
calculate out the corresponding
Figure BDA0003505429970000021
Value, substituted into the following equation:
Figure BDA0003505429970000022
Figure BDA0003505429970000023
the localization was judged by the proportion of lncRNA in the cytoplasm or nucleus.
Further, the cell membrane lysate containing NP-40 comprises the following components: 50mM Tris, 1.5mM MgCl 2 140mM NaCl, 0.5% NP-40, 1mM DTT and 1U/. Mu.LRNAsin.
Further, in the step (1), the specific operation of the cracking comprises: after being washed by PBS solution, the cells to be detected are resuspended in the cell membrane lysate containing NP-40 for lysis.
Further, in step (1), the cracking condition is 50min on ice.
Further, in step (4), the primer sequences for the endoplasmic reticulum reference GAPDH are shown as SEQ ID NO. 3 and SEQ ID NO. 4.
Further, in the step (4), the primer sequences used by the nuclear reference U6 are the sequences shown in SEQ ID NO. 5 and SEQ ID NO. 6.
Further, in the step (4), the reaction system of the qRT-PCR is: 2 × CHamQ univeral SYBR qRT-PCRMaster Mix 10 μ L, primer 10.4 μ L, primer 20.4 μ L, cDNA 1 μ L, RNase Free dH 2 Make up to 20. Mu.L of O.
Further, in step (4), the reaction procedure of the qRT-PCR is as follows: 95 ℃ 30sec,95 ℃ 10sec,60 ℃ 30sec,95 ℃ 15sec,60 ℃ 60sec,95 ℃ 15sec.
The invention discloses the following technical effects:
the invention utilizes the cell membrane lysate containing NP-40, can fully crack the cell membrane to release the cytoplasm content, and has weak nuclear membrane cracking effect; after centrifugation, the cytoplasm is located in the supernatant, while the uncleaved nuclei are located in the pellet, thus separating the cytoplasm from the nuclei. And respectively extracting cytoplasmic RNA and nuclear RNA from the separated cytoplasm and nucleus by adopting a Trizol extraction method, performing reverse transcription, and detecting target lncRNA, nuclear reference U6 and cytoplasmic reference GAPDH by qRT-PCR, thereby successfully detecting the subcellular localization of the lncRNA.
The GAPDH and U6 primers designed by the invention optimize the target specificity and GC content of the primers, reduce the probability of primer dimer and secondary structure, improve the detection sensitivity and specificity and ensure that the detection result is more reliable.
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In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings needed in the embodiments will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and it is obvious for those skilled in the art to obtain other drawings without creative efforts.
Fig. 1 is a subcellular distribution of LINC00707 in BGC cells.
Detailed Description
Reference will now be made in detail to various exemplary embodiments of the invention, the detailed description should not be construed as limiting the invention but as a more detailed description of certain aspects, features and embodiments of the invention.
It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. In addition, for numerical ranges in the present disclosure, it is understood that each intervening value, to the upper and lower limit of that range, is also specifically disclosed. Every intervening value, to the extent any stated or intervening value in a stated range, and every other stated or intervening value in that stated range, is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included or excluded in the range.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated herein by reference to disclose and describe the methods and/or materials in connection with which the documents are cited. In case of conflict with any incorporated document, the present specification will control.
It will be apparent to those skilled in the art that various modifications and variations can be made in the specific embodiments of the present disclosure without departing from the scope or spirit of the disclosure. Other embodiments will be apparent to those skilled in the art from consideration of the specification. The description and examples are intended to be illustrative only.
As used herein, the terms "comprising," "including," "having," "containing," and the like are open-ended terms that mean including, but not limited to.
Example 1 detection of subcellular distribution of LINC00707 in gastric carcinoma cells BGC
1. Preparation of cell membrane lysate
Cell membrane lysate is prepared, and the dosage and the preparation scheme of each reagent are shown in table 1.
TABLE 1
Figure BDA0003505429970000041
The solution can be stored in a refrigerator at 4 deg.C for a short time after being prepared. In carrying out the experiment, the following was further added thereto
The reagents are listed, namely the reagents are prepared as they are used.
Figure BDA0003505429970000042
2. Nuclear mass separation
Collect about 1.2X 10 6 Each BGC cell was washed three times in 1.5mL sterile, enzyme-free Ep tubes, centrifuged at 150 × g for 5 minutes with a pre-cooled sterile, enzyme-free PBS solution. Cells were resuspended in 200. Mu.L of cell membrane lysate, lysed on ice for 50 minutes, vortexed for 40 seconds with a vortexer, and then centrifuged at 3000 Xg for 2 minutes. Carefully pipette the supernatant into another 1.5mL sterile, enzyme-free Ep tube (to avoid aspiration of the pellet), which is the cytosolic fraction; the precipitate that remains is the nuclear fraction.
RNA extraction and cDNA Synthesis
(1) Respectively adding 1mLTRIzol reagent into the cell without nucleoplasm separation and the nucleus part and the cytoplasm part obtained by '2, nucleoplasm separation', and blowing and beating until complete lysis;
(2) Adding 0.2mL of chloroform into 1mL of LTRIzol, performing vortex oscillation for 15s until the materials are fully mixed, and standing for 3min;
(3) Centrifuging at 12000 Xg for 15min in a 4 deg.C precooled centrifuge;
(4) Transferring the water phase supernatant to a new 1.5mL sterile enzyme-free Ep tube, adding 400-500 mu L isopropanol according to the proportion of 1;
(5) Centrifuging at 12000 Xg for 10min in a 4 deg.C precooled centrifuge, and discarding the supernatant;
(6) Adding 1mL of precooled 75% ethanol into each tube, washing and precipitating, centrifuging for 10min at 7500 Xg in a centrifuge precooled at 4 ℃, and discarding the supernatant;
(7) Carefully absorbing and discarding excessive ethanol on the RNA precipitate, drying the precipitate for 2-3min until the product is transparent;
(8) The RNA precipitate was dissolved in 10-20. Mu.L of aqueous LDEPC and the RNA concentration was measured using a ultramicro spectrophotometer.
(9) RNA quality control: the RNA concentration and its absorbance values at wavelengths of 260nm, 280nm and 230nm, respectively, were measured using a spectrophotometer. The A260/A280 ratio of the RNA solution is a method for detecting RNA purity, and the range is generally 1.8-2.1. Below 1.8 indicates possible contamination and above 2.2 degradation. A relatively pure nucleic acid a260/a230 ratio of greater than 2.0;
(10) Synthesis of cDNA (PrimeScript) TM RT reagent Kit with gDNA Eraser (Perfect Real Time) from Takara: the reaction system and the reaction procedure are shown in Table 2.
TABLE 2
Figure BDA0003505429970000051
qRT-PCR detection
(1) Primer design
qRT-PCR primers were designed based on the LINC00707 sequence in NCBI, see Table 3. The qRT-PCR assay was performed using GAPDH as the cytoplasmic and U6 as the nuclear reference, and each sample was repeated 3 times. Separately detecting lncRNA, GAPDH and U6 from cDNA reverse-transcribed from total RNA, cytoplasmic RNA and nuclear RNA to obtain 9 sets of Ct value data including total lncRNA, total GAPDH, total U6, nuclear lncRNA, nuclear GAPDH, nuclear U6, cytoplasmic lncRNA, cytoplasmic GAPDH and cytoplasmic U6. The qRT-PCR detection reaction system and reaction procedure are shown in Table 4 and Table 5, respectively.
TABLE 3
Figure BDA0003505429970000061
TABLE 4 qRT-PCR reaction System (ChamQ Universal SYBR qRT-PCR Master Mix from Nodezak)
Figure BDA0003505429970000062
TABLE 5 qRT-PCR reaction procedure
Figure BDA0003505429970000063
5. Statistical analysis
Calculating octagon data of total GAPDH Ct-mean (total GAPDH Ct value), total U6 Ct-mean (total GAPDH Ct value), total GAPDH Ct-mean (total U6 Ct value), total U6 Ct-mean (total U6 Ct value), medium GAPDH Ct-mean (medium GAPDH Ct value), medium U6 Ct-mean (medium GAPDH Ct value), nuclear GAPDH Ct-mean (nuclear U6 Ct value), nuclear U6 Ct-mean (nuclear U6 Ct value) as delta Ct;
calculating four groups of [ substance GAPDH Ct value-mean (substance GAPDH Ct value) ] - [ total GAPDH Ct value-mean (total GAPDH Ct value) ], [ substance U6 Ct value-mean (substance GAPDH Ct value) ] - [ total U6 Ct value-mean (total GAPDH Ct value) ], [ nuclear GAPDH Ct value-mean (nuclear U6 Ct value) ] - [ total GAPDH Ct value-mean (total U6 Ct value) ], [ nuclear U6 Ct value-mean (nuclear U6 Ct value) ] - [ total U6 Ct value-mean (total U6 Ct value) ], as Δ;
calculate the corresponding
Figure BDA0003505429970000071
Value, substituted into the following equation:
Figure BDA0003505429970000072
Figure BDA0003505429970000073
since GAPDH is considered to be mainly present in cytoplasm and U6 is mainly present in nucleus, when GAPDH is more than 50% in cytoplasm and U6 is more than 50% in nucleus, it can be considered that nucleoplasm has been separated, and the higher its ratio, the better the nucleoplasm separation effect.
Calculating four groups of data (quality lncRNACt value-quality GAPDH Ct value), (total lncRNACt value-total GAPDH Ct value), (nuclear lncRNACt value-nuclear U6 Ct value), (total lncRNACt value-total U6 Ct value) as delta Ct;
calculating two groups of data (quality lncRNACt value-quality GAPDH Ct value) - (total lncRNACt value-total GAPDH Ct value), (nuclear lncRNACt value-nuclear U6 Ct value) - (total lncRNACt value-total U6 Ct value) as delta Ct;
calculate the corresponding
Figure BDA0003505429970000074
Values, substituted into the following equation:
Figure BDA0003505429970000075
Figure BDA0003505429970000076
the localization was judged by the proportion of lncRNA in the cytoplasm or nucleus.
Mean above represents taking the mean of three replicate wells.
Under the condition of successful nuclear and cytoplasmic separation, the proportion of lncRNA in cytoplasm exceeds 50%, and then the lncRNA is mainly distributed in the cytoplasm; if the proportion of lncRNA in the nucleus exceeds 50%, lncRNA is mainly distributed in the nucleus.
And (4) counting and analyzing results:
after statistical analysis, the proportion of GAPDH in cytoplasm is 66.581%, and the proportion of GAPDH in nucleus is 33.419%; the proportion of U6 in cytoplasm is 19.949%, and the proportion in nucleus is 80.051%. Indicating that BGC cells have been successfully isolated.
Calculating the proportion of LINC00707 in the cell nucleus and cytoplasm of the BGC to obtain that the proportion of LINC00707 in the cell nucleus of the BGC is 32.950% and the proportion of LINC00707 in the cytoplasm is 67.050%. It was therefore concluded that LINC00707 is mainly present in the cytoplasm of BGC cells, as shown in fig. 1.
The above-described embodiments are merely illustrative of the preferred embodiments of the present invention, and do not limit the scope of the present invention, and various modifications and improvements of the technical solutions of the present invention can be made by those skilled in the art without departing from the spirit of the present invention, and the technical solutions of the present invention are within the scope of the present invention defined by the claims.
Sequence listing
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Claims (8)

1. A method for detecting the location of lncRNA subcellular localization based on qRT-PCR technology is characterized by comprising the following steps:
(1) Separating nucleo-cytoplasmic: cracking the cell to be detected by using a cell membrane lysate containing NP-40 to obtain a nucleus part and a cytoplasm part;
(2) RNA extraction: extracting total RNA, nuclear RNA and cytoplasmic RNA from the cells without nucleoplasm separation and the nuclear part and the cytoplasmic part respectively by using a Trizol extraction method;
(3) cDNA Synthesis: reverse transcribing said nuclear RNA, said cytoplasmic RNA and said total RNA to synthesize nuclear cDNA, cytoplasmic cDNA and total cDNA, respectively;
(4) qRT-PCR detection: performing qRT-PCR detection by using GAPDH as a quality reference and U6 as a nuclear reference, and detecting lncRNA, GAPDH and U6 by using the nuclear cDNA, the quality cDNA and the total cDNA respectively to obtain 9 groups of Ct value data, namely total lncRNA, total GAPDH, total U6, nuclear lncRNA, nuclear GAPDH, nuclear U6, quality lncRNA, quality GAPDH and quality U6;
(5) Identification of lncRNA localization:
calculating four groups of data (substance lncRNA Ct value-substance GAPDH Ct value), (total lncRNA Ct value-total GAPDH Ct value), (nuclear lncRNA Ct value-nuclear U6 Ct value), and (total lncRNA Ct value-total U6 Ct value) as delta Ct;
calculating two groups of data (the medium lncRNA Ct value-the medium GAPDH Ct value) - (the total lncRNA Ct value-the total GAPDH Ct value), (the nuclear lncRNA Ct value-the nuclear U6 Ct value) - (the total lncRNA Ct value-the total U6 Ct value) as the Δ Ct;
calculate the corresponding 2 -△△Ct Values, substituted into the following equation:
cytoplasmic lncRNA ratio = mean [ cytoplasmic lncRNA 2 accordingly -△△Ct /(plasmid lncRNA corresponding 2) -△△Ct + Nuclear IncRNA corresponding to 2 -△△Ct )]×100％；
lncRNA ratio in nucleus = mean [ lncRNA of nucleus 2 -△△Ct /(plasmid lncRNA corresponding to 2) -△△Ct + Nuclear IncRNA corresponding 2 -△△Ct )]×100％；
The localization was judged by the proportion of lncRNA in the cytoplasm or nucleus.
2. The method for detecting the subcellular localization of lncRNA based on the qRT-PCR technology as claimed in claim 1, wherein the cell membrane lysate containing NP-40 comprises the following components: 50mM Tris, 1.5mM MgCl 2 140mM NaCl, 0.5% NP-40, 1mM DTT, and 1U/. Mu.L RNAsin.
3. The method for detecting the subcellular localization of lncRNA based on the qRT-PCR technology as claimed in claim 1, wherein in the step (1), the specific operation of lysis includes: after being washed by PBS solution, the cells to be tested are suspended in the cell membrane lysate containing NP-40 for lysis.
4. The method for detecting the subcellular localization of lncRNA based on the qRT-PCR technology of claim 3, wherein in the step (1), the lysis condition is 50min on ice.
5. The method for detecting the subcellular localization of lncRNA based on the qRT-PCR technology as claimed in claim 1, wherein in step (4), the primer sequences used by the internal control GAPDH are shown as SEQ ID NO. 3 and SEQ ID NO. 4.
6. The method for detecting the subcellular localization of lncRNA based on the qRT-PCR technology as claimed in claim 1, wherein in the step (4), the primer sequence used by the nuclear reference U6 is shown as SEQ ID NO. 5 and SEQ ID NO. 6.
7. The method for detecting lncRNA subcellular localization based on qRT-PCR technology according to claim 1, wherein in step (4), the reaction system of qRT-PCR is: 2 × CHamQ univeral SYBR qRT-PCR Master Mix 10 μ L, primer 1.4 μ L, primer 2.4 μ L, cDNA 1 μ L, RNase Free dH 2 O make up to 20. Mu.L.
8. The method for detecting lncRNA subcellular localization based on qRT-PCR technology according to claim 1, wherein in step (4), the reaction procedure of qRT-PCR is as follows: 95 ℃ 30sec,95 ℃ 10sec,60 ℃ 30sec,95 ℃ 15sec,60 ℃ 60sec,95 ℃ 15sec.
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CN106367526A (en) * 2016-11-04 2017-02-01 叶伟亮 Product for diagnosing prostatic cancer and application thereof
CN113717944A (en) * 2021-09-03 2021-11-30 江苏大学 miRNA13896 over-expressed engineered human umbilical cord mesenchymal stem cell-derived exosome and preparation method and application thereof

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