CN111235225A - Paraffin Embedding (FFPE) reference substance for detecting fusion gene RNA expression and preparation method and application thereof - Google Patents
Paraffin Embedding (FFPE) reference substance for detecting fusion gene RNA expression and preparation method and application thereof Download PDFInfo
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Abstract
The embodiment of the invention discloses a paraffin embedded (FFPE) reference substance for detecting fusion gene RNA expression and a preparation method and application thereof, wherein the method comprises the following steps: obtaining a cell line containing target fusion gene RNA expression; designing a paraffin-embedded RNA reference product; culturing and collecting cells; formalin fixation and paraffin embedding treatment are carried out on the collected cells; thus obtaining the FFPE reference substance for detecting the fusion gene RNA expression. The product and the method provided by the invention can well solve the problems that the form difference between an RNA solution reference substance and a clinical FFPE sample is huge, the RNA extraction process of FFPE cannot be monitored, the endogenous RNA expression profile of human cells cannot be reflected, and the product and the method cannot be well applied to RNA high-throughput sequencing such as RNA-seq and the like.
Description
Technical Field
The invention relates to the field of gene detection, in particular to a Formalin Fixed Paraffin Embedded (FFPE) reference product for detecting fusion gene RNA expression and a preparation method and application thereof.
Background
With the development of RNA detection technology, more and more fusion genes are gradually discovered in tumors. Gene fusion is a common feature of tumors, can promote the generation and development of tumors, and can be used as a molecular diagnosis and treatment target of tumors. Of all fusion mutations, 35% are kinase-associated mutations (n: 268), accounting for approximately one third. Since most of the kinase fusion mutations are continuously acquired in function, the kinase fusion mutation becomes a valid oncogenic mutation index and target. Fusion gene screening has become an important and necessary test marker before using targeted inhibitors to treat lung cancer. The national comprehensive cancer network clinical practice guideline, the European medical oncology society of cancer consensus and the Chinese expert consensus on molecular targeted therapy of advanced non-small cell lung cancer (NSCLC) are all clearly suggested: before receiving treatment, NSCLC patients should detect EGFR, ALK and ROS1 genes, determine corresponding treatment strategies according to the gene states, and recommend that the detection of the three genes be carried out simultaneously under the condition permission.
The gene detection needs reference substances for quality control. At present, few reports exist on RNA standard products, and an RNA quality control product (CN108085391A) of a lung cancer fusion gene detection kit capable of being stably stored adopts a method that fusion gene fragments are amplified by PCR and connected to plasmid to transform competent cells, pseudovirus solution is prepared, and RNA is extracted. This method has the following disadvantages: 1. the RNA solution has great difference with the common FFPE sample form of a tumor patient, and the form of a clinical sample cannot be well simulated; 2. the RNA solution cannot control the process of extracting RNA from FFPE; 3. RNA is derived from a few kinds of RNA expressed by plasmids, has great difference with total RNA expressed by human cells, cannot well simulate the state of fusion gene RNA existing in the total RNA in a clinical sample, and cannot be applied to RNA high-throughput sequencing such as RNA-seq.
Disclosure of Invention
Aiming at the technical problems, the embodiment of the invention provides an FFPE reference substance for detecting fusion gene RNA expression and a preparation method and application thereof, which can well solve the problems that an RNA solution reference substance has huge form difference with a clinical FFPE sample, the RNA extraction process of FFPE cannot be monitored, the endogenous RNA expression profile of human cells cannot be reflected, and therefore the FFPE reference substance cannot be well applied to RNA high-throughput sequencing such as RNA-seq and the like.
The first aspect of the embodiments of the present invention provides a method for preparing an FFPE reference substance for detecting RNA expression of a fusion gene, the method comprising the steps of:
obtaining of cell lines containing target fusion gene RNA expression:
a. integrating the fusion gene fragment into the genome of an immortalized human background cell line;
b. screening out monoclonal cell strains with fusion gene RNA expression and genetic stability;
c. determining the RNA expression intensity of the fusion gene of the monoclonal cell strain by adopting fluorescent quantitative reverse transcription PCR (qRT-PCR);
design of FFPE RNA reference: preparing a single fusion gene cell strain into an FFPE RNA reference product, or mixing a plurality of fusion gene cell strains according to a set quantity ratio to prepare the FFPE RNA reference product;
culturing and collecting cells;
formalin fixation and paraffin embedding treatment are carried out on the collected cells;
obtaining the FFPE reference substance for detecting the RNA expression of the fusion gene.
Alternatively, in the step of obtaining a cell line containing RNA expression of a fusion gene of interest, the fusion gene includes, but is not limited to, EML4-ALK, CCDC6-RET, SLC34A2-ROS1, TPM 3-NTRK 1, ETV6-NTRK3, and the like.
Alternatively, in step a of the obtaining of a cell line containing the expression of the target fusion gene RNA, the background cell line is a commercially immortalized cell line purchased from ATCC or other supplier; optionally, the background cell line is GM 12878;
alternatively, in the step of obtaining a of the cell line containing the target fusion gene RNA expression, a CRISPR/Cas9 system is adopted to design a specific guide RNA (guide RNA, gRNA) and an integration plasmid through homologous recombination, and the plasmid containing the fusion gene is integrated into a specific site of the cell genome. Wherein the plasmid used for integration comprises a homology arm, a target gene and a resistance selection gene. The site of integration can be selected as desired.
The background cell line of the invention is used as immortalized cell, which can be infinitely expanded to culture, thus being capable of large-scale stable production.
The fusion gene RNA is derived from human cell endogenous expression, and can well simulate the RNA state of a clinical sample.
Optionally, in the step of obtaining b, a cell line containing the target fusion gene RNA expression is obtained by selecting a genetically stable monoclonal cell line by using a monoclonal technique.
In one or more embodiments of the invention, the specific method is: when the cells reach a certain degree of confluence, the cells are digested and collected. The cells are accurately counted, and according to a method of dilution by multiple proportion, the living cells are diluted by a culture medium and the cells are mixed evenly. 10mL of the cell suspension was dispensed evenly into a 96-well plate. When the cell clone is formed, a monoclonal cell strain is obtained.
In one or more specific embodiments of the present invention, the cell lines obtained by the monoclonal technology include ALK-EML4, CCDC6-RET, SLC34A2-ROS1, TPM 3-NTRK 1, ETV6-NTRK3 and other fusion cell lines.
Optionally, in the step of obtaining b of the cell line containing the target fusion gene RNA expression, RT-PCR method is adopted to confirm the fusion gene RNA expression of the selected monoclonal cell strain.
In one or more embodiments of the present invention, total RNA in cells is extracted by Trizol reagent, first strand cDNA synthesis is performed by SuperScript reverse transcriptase from Thermo Fisher, and RT-PCR reaction is performed by Taq enzyme from Takara, which are all conventional molecular biology procedures and are not described herein.
Alternatively, in the step of obtaining b of the cell line containing the target fusion gene RNA expression, the cDNA sequence of the fusion gene was confirmed for the monoclonal cell line by Sanger sequencing.
In one or more embodiments of the invention, the Sanger sequencing service is provided by an organism.
In one or more embodiments of the present invention, qRT-PCR is performed using SuperReal Premix Plus (SYBR Green) kit from the Tiangen organism, and the procedures are described in the specification and will not be repeated herein.
Optionally, in the step of designing the FFPE RNA reference, according to the need, a single fusion gene cell line may be prepared into an FFPE wax block, or a plurality of fusion gene cell lines may be mixed according to a certain ratio to prepare an FFPE wax block, so that an FFPE reference including multiple fusion gene RNA expressions may be obtained.
Optionally, in the step of culturing and collecting cells, desired monoclonal cells are cultured, the cells are collected, the cells are counted, and a cell strain of a single fusion gene is used according to the requirement, or a plurality of cells are mixed according to a designed quantity ratio. The total number of cells required for each paraffin reference was 100X 106-500×106And (4) respectively. Preferably, the total number of cells per paraffin reference is 500X 106And (4) respectively.
Optionally, in the step of performing formalin-fixed paraffin embedding processing on the collected cells, a formalin solution is used as a fixing solution, paraffin is used as an embedding agent, and the fixed cells are embedded and sliced to obtain a paraffin-embedded reference substance; the specific method comprises the following steps:
A. fixing cells;
B. preparing and dehydrating cell clusters;
C. embedding and slicing the cells.
In one or more embodiments of the present invention, in step a, the cells are fixed with formalin solution.
Further, the fixed time is 12-72 h;
the cultured cells containing the target fusion gene expression are subjected to formalin-fixed paraffin embedding, so that the type of a clinical sample can be well simulated, and the quality control can be performed on the RNA extraction process of FFPE.
In one or more embodiments of the present invention, in the step B, dehydration treatment is performed using a Leica full-automatic dehydrator. The dehydration treatment may employ a conventional cell dehydration step.
In one or more embodiments of the present invention, in step C, the cell mass is dehydrated and then placed in an embedding mold, and an embedding machine is used to embed the cell mass; the embedded wax blocks were then sectioned, FFPE slices 3-30 μm thick.
The second aspect of the embodiment of the invention provides an FFPE reference substance prepared by the method for detecting the expression of the fusion gene RNA.
The third aspect of the embodiment of the invention provides application of the FFPE reference substance for detecting the fusion gene RNA expression in preparation of an RNA quality control product and/or a kit for detecting the fusion RNA expression.
Optionally, the FFPE reference is dewaxed, digested, and RNA extracted during use.
In dewaxing, dewaxing can be carried out by conventional techniques such as organic solvent dewaxing; when digesting, conventional technical means such as proteinase K can be adopted for digesting; when extracting RNA, an RNA kit is adopted for RNA extraction.
In various embodiments of the invention, the FFPE RNA extraction kit may be used to dewax, digest, and purify RNA, and the specific assay steps may be performed according to the instructions in the kit.
The fifth aspect of the embodiment of the invention provides an RNA quality control product, which comprises the FFPE reference product for detecting the RNA expression of the fusion gene.
The fifth aspect of the embodiment of the invention provides a kit for detecting fusion RNA expression, which comprises the FFPE reference substance or the RNA quality control substance for detecting fusion gene RNA expression.
The sixth aspect of the embodiment of the invention provides application of the RNA quality control product or the kit in gene detection.
Optionally, the RNA quality control product or the kit is applied to fusion gene detection.
Optionally, the fusion gene detection belongs to non-disease diagnosis and treatment.
Compared with the related technology known by the inventor, one technical scheme of the invention has the following beneficial effects:
in the technical scheme provided by the embodiment of the invention, fusion expression genes are integrated in the genome of the immortalized background cells, so that cells required for preparing FFPE RNA standard products can be stably obtained for a long time.
In the technical scheme provided by the embodiment of the invention, a monoclonal cell line containing target fusion gene RNA expression and stable heredity is obtained by using a cell monoclonal technology.
According to the technical scheme provided by the embodiment of the invention, the fusion gene is integrated into the genome of the human cell, so that the endogenous expression of any fusion gene in the human cell can be obtained.
According to the technical scheme provided by the embodiment of the invention, the cells are embedded by formalin-fixed paraffin to prepare the FFPE wax sheet, the FFPE wax sheet is consistent with the clinical FFPE sample form, and the RNA solution is used in the prior art.
In the technical scheme provided by the embodiment of the invention, the RNA is derived from cell endogenous expressed RNA, is consistent with the RNA form of a clinical FFPE sample, and can be applied to RNA high-throughput sequencing such as RNA-seq, while the prior art uses a few RNAs expressed by plasmids, which cannot reflect the expression profile of human cell endogenous RNA and can not be well applied to RNA high-throughput sequencing such as RNA-seq.
Drawings
The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate an embodiment of the invention and, together with the description, serve to explain the invention and not to limit the invention.
FIG. 1 shows Sanger sequencing verification of the cDNA sequence of the EML4-ALK fusion gene in example 1 of the present invention.
FIG. 2 is a graph showing the relative expression intensity of the EML4-ALK fusion gene RNA detected by qRT-PCR in example 1 of the present invention.
FIG. 3 is a photograph of an HE stained section in example 2 of the present invention.
FIG. 4 shows the RNA extraction of wax sheets from different positions of FFPE wax block in example 3.
FIG. 5 is an electropherogram of the PCR amplified fragment of the fusion gene in FFPE wax block in example 4 of the present invention.
Detailed Description
It is to be understood that the following detailed description is exemplary and is intended to provide further explanation of the invention as claimed. 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.
It is noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of exemplary embodiments according to the invention. As used herein, the singular forms "a", "an" and "the" are intended to include the plural forms as well, and it should be understood that when the terms "comprises" and/or "comprising" are used in this specification, they specify the presence of the stated features, steps, operations, and/or combinations thereof, unless the context clearly indicates otherwise.
Interpretation of terms:
fusion gene: it refers to a chimeric gene formed by connecting the coding regions of two or more genes end to end and placing them under the control of the same set of regulatory sequences (including promoter, enhancer, ribosome binding sequence, terminator, etc.).
Immortalized cells: unlike primary and passaged cells, immortalized cells have the ability to proliferate indefinitely. The immortalized cells can provide stable and uniform cell sources with consistent properties, are convenient to widely use, can reduce the cost of cell materials, are convenient to standardize and the like.
The number of the plurality of the particles in the present invention means 2 or more.
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
In the following examples, the reagents used were all analytical grade and were commercially available unless otherwise indicated. Experimental procedures not specifically identified herein are generally carried out under conventional conditions such as those described in the molecular cloning guidelines published by scientific Press, J. SammBruk et al, or under conditions recommended by the manufacturer. 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. In addition, any methods and materials similar or equivalent to those described herein can be used in the practice of the present invention.
Example 1
A preparation method of an FFPE reference substance for detecting fusion gene RNA expression comprises the following steps:
(1) obtaining cell lines containing expression of RNA of specific fusion genes
a. The fusion gene fragment is integrated into the genome of an immortalized human background cell line. Commercial immortalized cell lines were selected as background cell lines. In this example, GM12878 cell line was selected as the background cell line.
The specific method comprises the following steps: specific guide RNAs (guide RNAs, gRNAs) are designed aiming at the integration sites, and expression vectors of the guide RNAs are constructed. And constructing an integration plasmid which comprises elements such as a homologous left arm, a target gene, a resistance screening gene, a homologous right arm and the like. Typically, the length of the homology arms is 500-1000 bp. The expression vector for the guide RNA was transfected into the target cells and the integration plasmid was transfected again for 24 h. After 48h, the cell culture medium was changed to a medium containing antibiotics and the cells were selected.
b. And screening the monoclonal cell strain with stable fusion gene RNA expression and heredity by using a monoclonal technology, RT-PCR and Sanger sequencing technology.
The specific method comprises the following steps: cells screened with antibiotics were cultured to 80% confluence, digested and harvested. The cells are accurately counted, viable cells are diluted to 5 cells/ml by a method of dilution by multiple, and the cells are mixed evenly. 10mL of the cell suspension was dispensed evenly into a 96-well plate. When the cell clone is formed, a monoclonal cell strain is obtained.
The monoclonal cell strain obtained in this example has the fusion of EML4-ALK, CCDC6-RET, SLC34A2-ROS1, TPM 3-NTRK 1, ETV6-NTRK3 and the like.
In this example, total RNA in cells was extracted using Trizol reagent, first strand cDNA synthesis was performed using SuperScript reverse transcriptase from Thermo Fisher, and RT-PCR was performed using Taq enzyme from Takara. The amplification products of the RT-PCR were subjected to Sanger sequencing to confirm the cDNA sequence of the fusion gene, the Sanger sequencing service being provided by the organism. FIG. 1 shows that the fusion expression of the EML4 gene and ALK gene occurs through Sanger sequencing verification of the cDNA sequence of the fusion gene of EML4-ALK, which is completely consistent with the expectation. The identification results of other fusion genes are completely consistent with the expectation and are not displayed.
c. And determining the RNA expression intensity of the fusion gene of the monoclonal cell strain by adopting qRT-PCR.
In this example, the SuperReal PreMix Plus (SYBR Green) kit from Tiangen organisms was used to perform qRT-PCR with a BIORADCFX CONNECT fluorescent PCR instrument, according to the instructions. As a result of qRT-PCR shown in FIG. 2, the expression intensity of the EML4-ALK fusion gene was 21% of that of the reference gene TBP.
(2) Design of FFPE RNA reference: according to the needs, a single fusion gene cell strain can be prepared into the FFPE wax block, or a plurality of fusion gene cell strains can be mixed according to a certain quantity proportion to prepare the FFPE wax block, so that the FFPE reference substance containing the RNA expression of various fusion genes can be obtained.
(3) Cell culture and collection: and (3) culturing the cells by adopting an FBS culture medium, centrifugally collecting the cells after the culture is finished, counting the cells, and using a cell strain of a single fusion gene or mixing a plurality of cells according to a designed quantity proportion according to the requirement. The total number of cells required for each paraffin reference was 100×106-500×106And (4) respectively.
(4) Fixation of cells: the cells were fixed with 10% (v/v) formalin for 48 hours.
(5) Preparation and dehydration of cell mass: mixing the fixed cell suspension with the agarose gel solution with the same volume, and fixing and molding. And (4) performing dehydration treatment by using a Leica full-automatic dehydrator.
(6) Embedding and sectioning of cells: and (3) dehydrating the cell mass, putting the cell mass into an embedding mould, and embedding by adopting a come cotta CoreArcadia H + C embedding machine. The embedded wax block is placed on a come card RM2255 paraffin slicer for slicing, and the FFPE slice thickness is 3-30 μm, thus obtaining the FFPE reference product for detecting the fusion gene RNA expression.
Example 2
Staining sections with HE staining for uniformity of cell embedding
FFPE RNA wax blocks mixed with three fusion genes, namely EML4-ALK, SLC34A2-ROS1 and CCDC6-RET, prepared in example 1 are randomly subjected to HE staining on wax sheets at different positions, and cell embedding uniformity is observed.
The results are shown in FIG. 3, which shows that the cell dispersion uniformity is good.
Example 3
The FFPE RNA wax blocks mixed by three fusion genes of EML4-ALK, SLC34A2-ROS1 and CCDC6-RET prepared in the example 1 are randomly provided with wax sheets at different positions, and an FFPE RNA extraction kit is adopted
16 FFPEplus LEV RNA FFPE kit (Promega) is used for extracting RNA of the FFPE section, the concentration of the extracted RNA is measured by using the Qubit RNA HS Assay, the extracted RNA amount is shown in figure 4, and the extracted RNA amount of each FFPE section reaches the standard.
Example 4
Confirmation of expression of fusion gene involved in FFPE section by RT-PCR
The FFPE RNA wax block mixed with the prepared EML4-ALK, SLC34A2-ROS1 and CCDC6-RET three fusion genes is randomly subjected to wax sheet picking, the extracted RNA is subjected to reverse transcription and then the expression of the fusion genes is confirmed by RT-PCR, and as shown in figure 5, the result shows that the EML4-ALK, SLC34A2-ROS1 and CCDC6-RET genes in the FFPE wax sheet are all expressed.
Example 5
A kit for detecting fusion RNA expression comprises a primer for detecting a specific fusion gene, a Taqman probe, a quantitative PCR enzyme and a reaction buffer solution, and the FFPE RNA reference substance is used as a positive quality control substance of the kit.
The above examples are only intended to illustrate the technical solution of the present invention, but not to limit it; although the present invention has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some technical features may be equivalently replaced; and such modifications or substitutions do not depart from the spirit and scope of the corresponding technical solutions of the embodiments of the present invention.
Claims (10)
1. A preparation method of a formalin-fixed paraffin embedded (FFPE) reference substance for detecting fusion gene RNA expression is characterized by comprising the following steps:
obtaining of cell lines containing target fusion gene RNA expression:
a. integrating the fusion gene fragment into the genome of an immortalized human background cell line;
b. screening out monoclonal cell strains with fusion gene RNA expression and genetic stability;
c. determining the RNA expression intensity of the fusion gene of the monoclonal cell strain by adopting fluorescent quantitative reverse transcription PCR (qRT-PCR);
design of FFPE RNA reference: preparing a single fusion gene cell strain into an FFPE RNA reference product, or mixing a plurality of fusion gene cell strains according to a set quantity ratio to prepare the FFPE RNA reference product;
culturing and collecting cells;
formalin fixation and paraffin embedding treatment are carried out on the collected cells; thus obtaining the FFPE reference substance for detecting the fusion gene RNA expression.
2. The method of claim 1, further comprising: in the step of obtaining a cell line containing RNA expression of a fusion gene of interest, the fusion gene includes, but is not limited to, EML4-ALK, CCDC6-RET, SLC34A2-ROS1, TPM 3-NTRK 1, ETV6-NTRK 3;
alternatively, in step a of the obtaining of a cell line containing the expression of the target fusion gene RNA, the background cell line is a commercially immortalized cell line purchased from ATCC or other supplier; optionally, the background cell line is GM 12878;
optionally, in the step b of obtaining a cell line containing target fusion gene RNA expression, screening a monoclonal cell strain with fusion gene RNA expression and genetic stability by using a monoclonal technology, RT-PCR and sanger sequencing technologies;
alternatively, the cell line obtained by the monoclonal technique is subjected to RNA extraction and reverse transcription to amplify cDNA of the fusion gene, and then fusion of the gene is verified by Sanger sequencing.
3. The method of claim 1, further comprising: in the step of designing the FFPE RNA reference product, according to needs, a single fusion gene cell strain can be prepared into an FFPE wax block, or a plurality of fusion gene cell strains are mixed according to a certain proportion to be prepared into the FFPE wax block, so that the FFPE reference product containing multiple fusion gene RNA expressions can be obtained.
4. The method of claim 1, further comprising: in the step of culturing and collecting cells, desired monoclonal cells are cultured and collected for counting, and a cell line of a single fusion gene is used as required, or a plurality of cells are mixed in a designed number ratio.
5. The method of claim 1, further comprising: in the step of formalin fixation and paraffin embedding treatment of collected cells, formalin solution is used as fixing liquid, paraffin is used as embedding agent, and the fixed cells are embedded and sliced to obtain paraffin embedded reference products; the specific method comprises the following steps:
A. fixing cells;
B. preparing and dehydrating cell clusters;
C. embedding and slicing cells;
optionally, in the step a, formalin solution is adopted to fix the cells;
further, the fixed time is 12-72 h;
optionally, in the step B, dewatering is performed by using a dewatering machine or manually;
optionally, in the step C, the cell clusters are dehydrated and then placed into an embedding mold, and an embedding machine is adopted for embedding; the embedded wax blocks were then sectioned, FFPE slices 3-30 μm thick.
6. An FFPE reference product prepared by the method of any one of claims 1-5.
7. Use of the FFPE reference substance of claim 6 in the preparation of an RNA quality control and/or in the preparation of a kit for detecting expression of a fused RNA.
8. An RNA quality control product, which comprises the FFPE reference product for detecting the RNA expression of the fusion gene according to claim 6.
9. A kit for detecting expression of a fused RNA, comprising the FFPE reference substance of claim 6 or the RNA quality control substance of claim 8.
10. Use of the FFPE reference product of claim 6 or the RNA quality control product of claim 8 in genetic testing.
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| CN113106149A (en) * | 2021-04-26 | 2021-07-13 | 福建和瑞基因科技有限公司 | Preparation method of fusion gene detection reference substance and application of fusion gene detection reference substance |
| CN113881756A (en) * | 2021-11-01 | 2022-01-04 | 菁良基因科技(深圳)有限公司 | Quality control product for tumor fusion gene fluorescence in situ hybridization detection and preparation method thereof |
| CN117660604A (en) * | 2024-02-01 | 2024-03-08 | 广州迈景基因医学科技有限公司 | FFPE reference for NGS detection and preparation method and application thereof |
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