A translational omics reference standard and its preparation method

Abstract

The present invention discloses a translatomics standard product, a preparation method thereof, a screening method for a standard product cell line, and a related kit; the present invention prepares a translatomics standard product by using human liver cancer cell MHCC97H; the standard product has the advantages of small inter-generation differences and high stability, and provides a highly reproducible standard product for translatomics.

Description

Technical Field

[0001] The present invention belongs to the field of cellular translatomics, and particularly relates to a translatomics standard product and a preparation method thereof. Background Art

[0002] In recent years, with the rapid development of life omics research including genomics, transcriptomics, translatomics and proteomics, as well as the promotion of the cross - application of bioinformatics and big data science, a new medical concept and medical model, namely precision medicine, has emerged. The scientific paper "Translatomics: Methods and Applications" (Chemistry of Life, 2017, 37(1): 70 - 79) expounds that the broad - sense translatome refers to all elements directly involved in the translation process, including but not limited to ribosomes, translating mRNA (also known as RNC - mRNA), tRNA, regulatory RNAs (such as miRNA, lncRNA, etc.), nascent polypeptide chains, various translation factors, etc.; the narrow - sense translatome specifically refers to translating mRNA.

[0003] Life omics research is one of the most important means to reveal disease mechanisms and precision medicine. The quality of omics data is directly related to the accuracy of disease mechanisms and the effectiveness of precision medicine. However, at present, due to the lack of unified standards for the reference products selected by each laboratory, diverse sample processing methods, diverse measurement platforms and inaccurate analysis techniques, the repeatability and experimental performance of cross - platform and cross - laboratory data are poor. The Universal Human Reference RNA (UHRR) is a mixed RNA of 10 cancer cell lines including HeLa, and its instability is well - known. In fact, in the comparison between UHRR batches, the Pearson correlation only reaches R 2 = 0.9478, which indicates that this instability exists even within a short production cycle. This variation is expected to multiply within a long production cycle and is insufficient to evaluate the reproducibility of the next - generation sequencing technology that is evolving with increasing depth and resolution. The Hela cells used as reference products in the past have been widely debated due to their strong intra - species and inter - species contamination. Some studies have shown that by culturing the Hela cell lines used in different laboratories together and continuously passaging them 50 generations, using the latest quantitative transcriptomics and other omics technologies, combined with molecular cytology experiments, it is found that the Hela cells with the same name have significant biological variations at the phenotypic and various molecular levels. Therefore, selecting a reference product with robustness and consistency at multiple omics levels is the key to ensuring the stable output of omics data. Summary of the Invention

[0004] This study provides a translational omics standard product, a preparation method thereof, a screening method for a standard product cell line, and a kit thereof through examples, thereby obtaining a highly stable translational omics standard product.

[0005] To achieve the above object, the present invention is implemented according to the following technical solutions:

[0006] A translational omics standard product, wherein the standard product is total RNC-mRNA extracted from the MHCC97H cell line.

[0007] Preferably, the total mRNA is prepared by the following method: Step 1, culture MHCC97H cells; Step 2, isolate RNC from the cells; Step 3, isolate RNC-mRNA from the RNC.

[0008] The present invention also provides a kit, which contains the translational omics standard product according to any one of claims 1 or 2.

[0009] The present invention also provides a preparation method for a translational omics standard product; the method includes the following steps:

[0010] Step 1, culture MHCC97H cells;

[0011] Step 2, isolate RNC from the cells;

[0012] Step 3, isolate RNC-mRNA from the RNC.

[0013] Preferably, the specific operation of step 2 is as follows:

[0014] 1. When the cells grow to confluence in a 75 cm 2 culture flask, add cycloheximide to a final concentration of 100 μg / mL, and incubate in an incubator for 15 min;

[0015] 2. Discard the culture supernatant, place the culture flask on ice, and wash twice with pre-cooled PBS; in the present invention, unless otherwise specified, pre-cooling means placing the reagent in an ice-water mixture until its temperature drops to the same as that of the ice-water mixture.

[0016] 3. Completely discard the PBS, add 2 mL of pre-cooled RB lysis buffer, and lyse on ice for 30 min;

[0017] 4. Scrape the cells with a cell scraper, and transfer the lysate to a pre-cooled EP tube;

[0018] 5. Centrifuge at 13200 rpm at 4 °C for 10 min;

[0019] 6. Add 15 mL of pre-cooled 30% sucrose solution to an ultracentrifuge tube;

[0020] 7. Transfer the supernatant in Step 5 into an ultracentrifuge tube.

[0021] 8. Ultracentrifuge at 42500 rpm and 4 °C for 5 h.

[0022] 9. Discard the supernatant, and carefully wash the surface of the RNC precipitate with pre-cooled PBS twice to obtain RNC.

[0023] Preferably, the specific operation of Step 3 is as follows:

[0024] 1. Add 1 mL of Trizol to the RNC precipitate obtained in Step 2, add 0.2 mL of chloroform to every 1000 μL of Trizol, shake vigorously and mix well for 15 s, and let stand at room temperature for 3 min.

[0025] 2. Centrifuge at 12000×g and 4 °C for 15 min to separate the sample into three layers.

[0026] 3. Aspirate the upper aqueous phase and transfer it to a new 1.5 mL EP tube, and do not aspirate the liquid in the middle layer during the operation.

[0027] 4. Add 800 μL of isopropanol and invert to mix well.

[0028] 5. Let stand at -20 °C for 8 h.

[0029] 6. After 8 h, centrifuge at 12000×g and 4 °C for 30 min to remove the supernatant.

[0030] 7. Add 1 mL of 75% ethanol; the ethanol needs to be pre-cooled at -20 °C for 30 min in advance.

[0031] 8. Centrifuge at 7500×g and 4 °C for 5 min to remove the supernatant.

[0032] 9. Repeat Steps 7) and 8) once.

[0033] 10. Remove the residual ethanol, open the tube cap, and air dry for 5 min.

[0034] 11. Dissolve with about 30 - 60 μL of RNase Free pure water according to the particle size. This step obtains the RNC-mRNA standard product, which is stored in a -80 °C refrigerator.

[0035] The present invention also provides a method for screening a translational omics standard cell line, including the following steps:

[0036] Step 1: Culture the target cells to be screened. Specifically: Culture the cells using DMEM complete medium containing 10% fetal bovine serum, 1% double antibody, and 0.5% ciprofloxacin. When the cells reach 90% density, digest them with trypsin for subculture. Continuously collect cells from 8 to 10 passages, and collect 8×10 6 cells each time for standby;

[0037] Step 2: Isolate RNC from the cells. Specifically

[0038] 1. When the cells cover a 75 cm 2 culture flask, add cycloheximide to a final concentration of 100 μg / mL and incubate in an incubator for 15 min;

[0039] 2. Discard the culture supernatant, place the culture flask on ice, and wash it twice with pre-cooled PBS;

[0040] 3. Completely discard the PBS, add 2 mL of pre-cooled RB lysis buffer, and lyse on ice for 30 min;

[0041] 4. Scrape the cells with a cell scraper and transfer the lysate to a pre-cooled EP tube;

[0042] 5. Centrifuge at 13200 rpm at 4°C for 10 min;

[0043] 6. Add 15 mL of pre-cooled 30% sucrose solution to an ultracentrifuge tube;

[0044] 7. Transfer the supernatant from step 5 to the ultracentrifuge tube;

[0045] 8. Ultracentrifuge at 42500 rpm at 4°C for 5 h;

[0046] 9. Discard the supernatant, carefully wash the surface of the RNC pellet twice with pre-cooled PBS; Obtain RNC;

[0047] Step 3: Isolate RNC-mRNA from RNC. The specific steps are as follows:

[0048] 1. Add 1 mL of Trizol to the RNC pellet obtained in step 2. For every 1000 μL of Trizol, add 0.2 mL of chloroform, shake vigorously for 15 s, and let it stand at room temperature for 3 min;

[0049] 2. Centrifuge at 12000×g at 4°C for 15 min to separate the sample into three layers;

[0050] 3. Pipette the upper aqueous phase and transfer it to a new 1.5 mL EP tube. Do not pipette the liquid in the middle layer during the operation;

[0051] 4. Add 800 μL of isopropanol and invert to mix;

[0052] 5. Place it at -20°C and let it stand for 8 h;

[0053] 6. After 8 h, centrifuge at 12,000×g at 4°C for 30 min to remove the supernatant;

[0054] 7. Add 1 mL of 75% ethanol; the ethanol needs to be pre-cooled at -20°C for 30 min in advance;

[0055] 8. Centrifuge at 7,500×g at 4°C for 5 min to remove the supernatant;

[0056] 9. Repeat steps 7) and 8) once;

[0057] 10. Remove the residual ethanol, open the tube cap, and air-dry for 5 min;

[0058] 11. Dissolve it by adding about 30 - 60 μL of RNase Free pure water according to the particle size, and the total RNC-mRNA sample is obtained in this step;

[0059] Step 4. Evaluation of the quantitative stability of the translatome: Sequence the RNC-mRNAs of different passages of each cell line obtained in step 3 for qualitative and quantitative detection; perform a correlation analysis on the expression levels of the RNC-mRNA groups between different passages of the same cell line to obtain the Pearson correlation coefficient; compare the correlation coefficients between different cell lines, and the cell line with the highest correlation coefficient is selected as the cell line for preparing the translatome standard.

[0060] In the screening method described in the present invention, the correlation coefficients of multiple cell lines can be directly compared horizontally by the method of step 4 above, or a primary screening can be performed based on the correlation of transcriptome data to obtain a cell line with relatively stable transcriptome level; then, the correlation coefficient analysis of the translatome between passages of the selected relatively stable target cell line is performed, and the cell line with the highest correlation coefficient is selected as the standard cell line.

[0061] Preferably, the target cells for screening in step 1 are human liver cancer cells MHCC97H, HCCLM6, and HCCLM3.

[0062] The beneficial effects of the present invention are as follows: The present invention continuously collects five cell lines, namely, the human hepatocellular carcinoma cell lines MHCC97H, HCCLM3, HCCLM6, the human lung cancer cell line A549, and the human cervical cancer cell line Hela, and performs transcriptome detection. After excluding the A549 and Hela cells with poor transcriptome stability, the translatome sequencing of the MHCC97H, HCCLM3, and HCCLM6 hepatocellular carcinoma cells is then carried out. The cell line MHCC97H with the highest translatome stability during cell passage is evaluated and obtained. Based on this, the cell line with the highest stability is selected as the collection object of the candidate reference substance, and a reference cell line with high robustness at the translatomics level is screened out as a widely promoted standard product. And based on this, a translatome library construction and analysis process for mildly degraded samples is developed to increase the robustness of such samples. The present invention demonstrates a translatome-stable human cell line across multiple passages and its corresponding standard product, which is suitable as a material for long-term production of reference translatome standard substances. BRIEF DESCRIPTION OF THE DRAWINGS

[0063] In order to clearly show the specific implementation manners of the present invention and certain detection techniques adopted in the experiments, the implementation manners and the techniques adopted will be described below, mainly introduced in the form of drawings.

[0064] Figure 1 . Quantitative stability evaluation of the transcriptomes of five cell lines at different passages. Figures A - E are respectively the quantitative correlation analysis of the transcriptomes among different passages of the five cell lines MHCC97H, HCCLM6, HCCLM3, Hela, and A549. The horizontal and vertical coordinates represent different cell passages; Figure F is the technical replicates of three transcriptome sequencings of the 3rd, 5th, 7th, and 9th generation cells of the MHCC97H cells respectively.

[0065] Figure 2. Multi - center and multi - spatio - temporal sequencing stability assessment of the MHCC97H transcriptome. Two batches of RNA from different generations of MHCC97H cell lines were obtained at different times (May and December 2021), subjected to transcriptome library construction and sequencing, and quantitative correlation analysis was carried out. Among them, Figure A is the quantitative correlation analysis of transcriptome sequencing in December 2021; Figure B is the comparative analysis of quantitative correlation of corresponding samples between December and May. After extracting the RNA of different generations of MHCC97H cell lines, it was divided into two parts and given to two experimentalists with 2 years of high - throughput sequencing experience respectively for transcriptome library construction and sequencing. Among them, Figure C is the result of quantitative correlation analysis of transcriptome sequencing of different generations of MHCC97H obtained under the operation of Experimenter 2; Figure D is the result of comparative analysis of quantitative correlation of transcriptome sequencing of MHCC97H between Experimenter 1 and Experimenter 2. After extracting the RNA of different generations of MHCC97H cell lines and dividing it into two parts, the RNA samples of MHCC97H were sent to Chengqi Biotech and Saizhe Gene respectively. Figures E and F are the results of quantitative correlation analysis of transcriptome sequencing of the two commercial companies, and the horizontal and vertical coordinates are cell generations. Different sequencing data alignment algorithms were used to analyze the sequencing data of different generations of the MHCC97H transcriptome of the same batch to obtain quantitative correlation analysis data; Figure G is the quantitative correlation analysis of the same sample by different laboratories; Figure H uses the Bowtie2 algorithm, and Figure I uses the Hista algorithm. After adopting two different ribosomal RNA removal procedures respectively, transcriptome sequencing was carried out. Figure J is the result of correlation analysis of the MHCC97H transcriptome data measured after removing ribosomal RNA by the Ribominus method, and Figure K is the result of comparative analysis of quantitative correlation of MHCC97H transcript sequencing between the PolyA + mRNA enrichment method and the Ribominus method.

[0066] Figure 3 . Evaluation of the library construction method for natural degradation RNA samples. Figure A: RNA degradation of different generations of MHCC97H cells caused by simulated environmental exposure, and the obtained degraded RNA was subjected to agarose gel electrophoresis; Figures B - D used different algorithms (FANSe, bowtie2, and Hista) to conduct a comparative analysis of quantitative correlation of sequencing data of degraded RNA samples and non - degraded RNA samples of different generations of MHCC97H constructed by the polyA + mRNA enrichment method. Normal represents non - degraded RNA samples, and degradation represents degraded samples.

[0067] Figure 4Quantitative stability assessment of the translation group of MHCC97H. Samples were continuously collected from the 6th generation to the 12th generation of the MHCC97H cell line. Two independent replicate samples were designed for each generation, and RNC-mRNA extraction was performed, resulting in a total of 14 samples. Translational group sequencing was carried out, and pairwise translational group quantitative data between samples were obtained after data analysis. Pearson correlation analysis was performed to obtain the correlation coefficient R, which is shown in the grid.

[0068] Figure 5 Quantitative stability assessment of the translation group of HCCLM3. Samples were continuously collected from the 6th generation to the 12th generation of the HCCLM3 cell line. Two independent replicate samples were designed for each generation, and RNC-mRNA extraction was performed, resulting in a total of 14 samples. Translational group sequencing was carried out, and pairwise translational group quantitative data between samples were obtained after data analysis. Pearson correlation analysis was performed to obtain the correlation coefficient R, which is shown in the grid.

[0069] Figure 6 Quantitative stability assessment of the translation group of HCCLM6. Samples were continuously collected from the 6th generation to the 12th generation of the HCCLM6 cell line. Two independent replicate samples were designed for each generation, and RNC-mRNA extraction was performed, resulting in a total of 14 samples. Translational group sequencing was carried out, and pairwise translational group quantitative data between samples were obtained after data analysis. Pearson correlation analysis was performed to obtain the correlation coefficient R, which is shown in the grid.

[0070] Figure 7 Quantitative stability assessment of the proteome of MHCC97H. Figure A shows the results of the quantitative correlation analysis of the translational group sequencing of different generations of MHCC97H; Figures B / C show the results of the quantitative correlation analysis of the proteomics collected in the DDA and DIA modes for different generations of MHCC97H cells; Figures D / E show the results of the quantitative correlation analysis of the proteome in three independent experiments for the third-generation MHCC97H cells in the DDA and DIA collection modes.

[0071] Figure 8 Quantitative correlation analysis of the proteome of MHCC97H in different laboratories. Figure A shows the mass spectrometry, chromatographic models, high-performance liquid chromatography column and analytical column specifications used by four laboratories; Figure B shows the isoelectric point distribution map of the identified proteins in different laboratories and the P-value results obtained from pairwise KS analysis; Figures C / D show the correlation analysis results of the identified proteins in the DDA and DIA modes for the same sample in different laboratories.

[0072] Specific implementation methods

[0073] Specific embodiments of the present invention are explained by way of examples. Except for the techniques used in the detection which do not impose any form of limitation on the present invention, some of the solutions in the described embodiments are part of the embodiments of the invention. Embodiments obtained by ordinary technical operators in the art without creative achievements fall within the protection scope of the present invention.

[0074] Example 1

[0075] Main reagents and formulations

[0076] Cell lines

[0077] Human liver cancer cells MHCC97H, HCCLM6, and HCCLM3 were purchased from the Institute of Biomedical Sciences, Fudan University; MHCC97H cells are common and easily obtainable cells, and the sources of this cell line have been disclosed in multiple authorized patents (CN107523544B, CN102813643B). Human cervical cancer Hela cells and human non-small cell lung cancer cells A549 were purchased from the American Type Culture Collection.

[0078] Cell culture reagents

[0079] Dulbecco's modified eagle medium (DMEM) medium, from Life technology, USA;

[0080] Fetal bovine serum (FBS), from Life technology, USA;

[0081] Penicillin-streptomycin (PS), from Jiebeisi Biotechnology Co., Ltd., Guangzhou;

[0082] Trypsin, from Amresco, USA;

[0083] Ciprofloxacin (CIP), from Guangzhou Nanxin Pharmaceutical Co., Ltd.;

[0084] Dimethylsulfoxide (DMSO), from Sigma, USA;

[0085] T25, T75, and T175 cell culture flasks, from Becton Dickison, USA;

[0086] L-Glutamine, from Sigma, USA;

[0087] Main instruments

[0088] Analytical balance, Syngene, UK;

[0089] MilliPore pure water system, MilliPore, USA;

[0090] Laminar flow hood, Taosu Jing'an Co., Ltd., Jiangsu, China;

[0091] Small high-speed refrigerated centrifuge, Eppendorf, Germany;

[0092] Ice maker, Sanyo, Japan;

[0093] Optima L-XP ultracentrifuge, Beckman, USA;

[0094] BGIseq500 / 2000 sequencer, MGI Tech Co., Ltd., China;

[0095] RNC extraction reagent and formula

[0096] 1. RB buffer: 5 mL of 1 M HEPES, 2.5 mL of 1.5 M MgCl2, 25 mL of 2 M KCl, 212.5 mL of ultrapure water. Autoclave at 121 °C for 1 h by wet method. After cooling to room temperature, add 2.5 mL of 200 mM DTT and 2.5 mL of 10 mg / mL cycloheximide, and adjust the pH to 7.4 with KOH.

[0097] 2. RB lysis solution: RB buffer containing 1% Triton X-100.

[0098] 3. 30% sucrose solution: 30 g of sucrose, 100 mL of RB buffer.

[0099] Experimental method

[0100] Cell culture

[0101] Cells were cultured in DMEM complete medium containing 10% fetal bovine serum, 1% double antibody, and 0.5% ciprofloxacin. When the cells reached 90% density, they were digested with trypsin for subculture. A total of 10 passages of cells were collected continuously, and 8×10 6 cells were collected each time for translatome sequencing. All cells involved in this patent have been tested for mycoplasma and identified by cell STR, and no mycoplasma contamination or cell cross-contamination was found.

[0102] RNC isolation

[0103] For the specific method, please refer to references [1, 2], and the following specific steps are also available:

[0104] 1. When the cells reached confluence in a 75 cm 2Add cycloheximide at a final concentration of 100 μg / mL to the culture flask and incubate in an incubator for 15 min.

[0105] 2. Discard the culture supernatant, place on ice, and wash twice with pre-cooled PBS.

[0106] 3. Completely discard the PBS, add 2 mL of pre-cooled RB lysis buffer, and lyse on ice for 30 min.

[0107] 4. Scrape the cells with a cell scraper and transfer the lysate to a pre-cooled EP tube.

[0108] 5. Centrifuge at 13200 rpm at 4 °C for 10 min.

[0109] 6. Add 15 mL of pre-cooled 30% sucrose solution to the ultracentrifuge tube.

[0110] 7. Carefully transfer the supernatant from step 5 to the ultracentrifuge tube.

[0111] 8. Ultracentrifuge at 42500 rpm at 4 °C for 5 h.

[0112] 9. Discard the supernatant and carefully wash the surface of the RNC pellet twice with pre-cooled PBS.

[0113] RNC-mRNA Extraction

[0114] Add 1 mL of pre-cooled total RNA extraction reagent to the RNC pellet obtained in the previous step and extract RNC-mRNA according to the instructions. Add 1 mL of pre-cooled Trizol (total RNA extraction reagent), mix well by vigorous shaking. The specific extraction procedure is as follows:

[0115] 1. Add 0.2 mL of chloroform to every 1000 μL of Trizol, mix vigorously (can be vortexed) for 15 s, and let stand at room temperature for 3 min. It can be observed that the sample starts to separate into layers.

[0116] 2. Centrifuge at 12000×g at 4 °C for 15 min to separate the sample into three layers.

[0117] 3. Carefully aspirate the upper aqueous phase, about 600 μL, and transfer it to a new 1.5 mL EP tube, taking care not to aspirate the liquid in the middle layer during the operation.

[0118] 4. Add 800 μL of isopropanol and mix by inverting.

[0119] 5. Let stand at -20 °C for 8 h.

[0120] 6. After 8 h, centrifuge at 12000×g at 4 °C for 30 min to remove the supernatant.

[0121] 7. Add 1 mL of 75% ethanol (pre-cooled at -20 °C for 30 min in advance);

[0122] 8. Centrifuge at 7500×g for 5 min at 4 °C to remove the supernatant;

[0123] 9. Repeat steps 7) and 8) once;

[0124] 10. Remove the residual ethanol, open the tube lid, and air-dry for 5 min;

[0125] 11. Dissolve with approximately 30 - 60 μL of RNase Free pure water according to the size of the pellet. The RNC standard is obtained in this step and stored in a -80 °C refrigerator.

[0126] RNA Extraction and Transcriptome Sequencing Library Construction

[0127] It is carried out according to the library construction and sequencing process of the human poly-A mRNA standard transcriptome. The specific experimental steps can be referred to the published literature [1, 2] of the applicant.

[0128] The extraction process of total RNA is as follows: When the cells grow to confluence in a 75 cm 2 culture flask, discard the culture supernatant, place on ice, and wash twice with pre-cooled PBS. Discard all PBS completely, add 5 mL of pre-cooled Trizol (total RNA extraction reagent) for extraction. The specific steps can be referred to the Trizol instruction manual.

[0129] The process of enriching total mRNA uses the Novoprotein poly-A mRNA enrichment kit (VAHTS mRNA Caputre Beads) to enrich poly-A mRNA. The specific steps are shown in the instruction manual:

[0130] Degraded RNA Sample Transcriptome Library Construction

[0131] Use the Novoprotein Ribo-off rRNA depletion kit (Human / Mouse / Rat) removal kit to construct a library for the degraded RNA sample. The specific steps can be referred to the product instruction manual.

[0132] The library is constructed using the MGI transcriptome library construction kit. The PE150 sequencing is carried out using the BGIseq2000 high-throughput sequencing platform. The main sequencing data is aligned to the human transcriptome reference sequence using FANSe3 with the parameters of -L80 -E5 -I0 -S14 -B1 -U0.

[0133] Analysis Parameters of Different Mapping Algorithms Appearing in this Patent

[0134]

[0135] Protein Extraction

[0136] Protein extraction was carried out according to the protein extraction instructions of Beyotime's SDS protein lysis buffer for cell samples. The specific operation method is as follows:

[0137] 1. Dissolve the SDS lysis buffer and mix well. Take an appropriate amount of lysis buffer and add PMSF within a few minutes before use to make the final concentration of PMSF 1 mM;

[0138] 2. Remove the cell culture medium and wash once with PBS;

[0139] 3. According to the number of cells, add lysis buffer at a ratio of 150 - 250 μL of lysis buffer per million cells. Pipette several times to make the lysis buffer fully contact with the cells;

[0140] 4. Lyse on ice for 30 min. Vortex for 15 s every 10 minutes;

[0141] 5. Centrifuge at 17000×g, 4℃ for 30 minutes, and aspirate the supernatant as the protein standard. Store at -80°C refrigerator.

[0142] If necessary, a BCA protein concentration quantification kit can be used to detect the protein concentration.

[0143] Enzymatic digestion by Filter-aided sample preparation (FASP)

[0144] 1. Add an appropriate volume of protein denaturant to each tube of 200 μg protein sample to make the final concentration of urea greater than 6 M. If there is visible precipitation in the liquid at this time, sonicate at 35% power for 20 s, pausing every 5 s;

[0145] 2. Add 50 mM DTT and place in a 37°C water bath for 1 h;

[0146] 3. Add 100 mM IAA and let stand at room temperature for 30 min;

[0147] 4. Transfer the reduced and alkylated protein sample to a 30 kDa ultrafiltration tube and centrifuge at 4℃, 12000×g for 20 min;

[0148] 5. Add 100 μL of 8 M urea, centrifuge at 4℃, 12000×g for 20 min, and repeat this step once;

[0149] 6. Add 100 μL of 50 mM TEAB, centrifuge at 4℃, 12000×g for 20 min, and repeat this step four times;

[0150] 7. After adding 50 μL of 50 mM TEAB, trypsin was added, and the ratio of protein amount to trypsin amount was 1:30.

[0151] 8. The ultrafiltration tube was placed in a clean sleeve, the tube mouth was sealed with sealing film, and it was incubated in a 37°C incubator for 12 h.

[0152] 9. The peptide segments were collected by centrifugation at 12000×g for 20 min at 4°C.

[0153] 10. The peptide segment samples were freeze-dried and stored in a -80°C refrigerator.

[0154] Reversed-phase liquid chromatography combine massspectrum, RPLC-MS

[0155] 1. The peptide segments were dissolved with 10 μL of Solution A, and a Durashell C 18 (L) (2.1 mm × 50 mm) (particle size: 3 μm; pore size: ) Eksigent chromatograph was used for pre-separation of components. The chromatographic separation time was 45 min, and six components were separated from each sample. The specific gradient of the mobile phase separation is shown in the following table:

[0156]

[0157] 2. The pre-separated peptide segment components were freeze-dried, the peptide segment powder was dissolved with 25 μL of Solution A, centrifuged at 12000 ×g for 20 min at 4°C, and 10 μL of the sample was taken for MS identification.

[0158] 3. The specific parameters of the Orbitrap Fusion mass spectrometer were set as follows: the cycle time was set to 3.5 s, the resolution of MS1 was 120K, the scanning range of MS1 was 350 - 1550; the resolution of MS2 was set to 15K, the ion fragmentation mode was HCD, dynamic exclusion was 45 s, n = 1, the AGC threshold was 5e4, and the maximum injection time was 90 ms; a raw file was obtained, and this file was the original data of the proteome.

[0159] Database search

[0160] Protein identification and retrieval software Maxquant 1.6.2 was used for protein database searching for qualitative and quantitative analysis. The database used and the database searching parameters were as follows: the tolerance of MS1 was 10 ppm, the tolerance of secondary fragments was 0.02 Da, the maximum number of missed cleavages allowed was 2, Carbamidomethyl (cysteine) was a fixed modification, methionine oxidation (methionine) and acetylation (protein N-terminus) were variable modifications. For protein identification control, the false discovery rate (FDR) at the peptide level was <0.01, and the FDR at the protein level was <0.01. The specific peptides were at least greater than 1.

[0161] References: If there are any incomplete parts in the methods used in the present invention, the following two references can be referred to.

[0162] 1. Wang, T., et al., Translating mRNAs strongly correlate to proteins in a multivariate manner and their translation ratios are phenotype specific. Nucleic Acids Res, 2013. 41(9): p. 4743-54.

[0163] 2. Li, D., et al., Optimal Settings of Mass Spectrometry Open Search Strategy for Higher Confidence. J Proteome Res, 2018. 17(11): p. 3719-3729.

[0164] Results and Discussion of Example 2

[0165] MHCC97H can be used as a cell line with a stable source of transcriptome standard

[0166] Discovery and screening of potential stable reference cell lines

[0167] Five commonly used cell lines, MHCC97H, HCCLM6, HCCLM3, HeLa and A549, were detected in the present invention. For each cell line, the present invention carried out culturing for 8-11 generations and sampled from each generation. Total RNA was extracted from each sample, and the RNA quality was checked by electrophoresis to verify that they were not degraded. Then polyA+ mRNA was sequenced and quantified using the RPKM method. The cross-correlation analysis among them showed that the transcriptome of MHCC97H was the most stable, Pearson R 2 = 0.966 - 0.995 ( Figure 1A). The consistency of the other two hepatocellular carcinoma cell lines was relatively low (R 2 which could be as low as 0.947 and 0.921 respectively, Figure 1 B–C). The consistency between generations of HeLa and A549 cells was even lower (R 2 which were as low as 0.899 and 0.846 respectively, Figure 1 D–E). Specifically, this deviation includes biological deviation (the inconsistency of the transcriptome between generations) and experimental error (errors generated by library construction, sequencers, and data processing algorithms). The present invention also examined the experimental error by independently sequencing the same MHCC97H RNA samples from different generations three times and obtained an average R 2 = 0.98 Pearson correlation ( Figure 1 F). This indicates that Figure 1 the deviation shown in A can be almost explained by experimental error, that is, the biological deviation of MHCC97H is almost negligible. In contrast, the lower limit of R 2 of other tested cell lines decreased with the passage of generations ( Figure 1 B–E), indicating that their biological differences increased significantly with the passage of generations and thus they are not ideal candidates for standards.

[0168] Assessment of the transcriptome stability of MHCC97H

[0169] A good reference standard should be easy to produce and supplemented with a robust standard processing protocol which, ideally, can produce always the same results. The main steps introducing deviation are cell culture, mRNA enrichment, library construction / sequencing, and data processing algorithms. First, two batches of the MHCC97H cell line were passaged in May and December 2021 respectively. The generations of gene expression were similar, indicating that the cell cultures were consistent. Second, the present invention tested the robustness of the process by different experimenters and in different laboratories. The present invention used the same series of total MHCC97H RNA as the starting material and asked another experimenter to independently construct libraries using another batch of library construction kits. The results ( Figure 2 C / D) were almost identical to those of the previous experimenter ( Figure 1 A). In addition, the present invention also sent 4 samples to two commercial sequencing service providers more than 1000 kilometers away. Chi-Biotech Co., Ltd. was equipped with an MGISEQ-2000 sequencer and Sagene Co., Ltd. was equipped with an Illumina NovaSeq-6000 sequencer. They used Illumina kits to construct libraries and operated the sequencers according to the manufacturer's instructions. R 2The coefficients reached 0.959 - 0.981 and 0.941 - 0.981( Figure 2 E - F). The quantitative correlation analysis was respectively performed on the sequencing data corresponding to the two commercial companies and the data generated by Jinan University. The quantitative correlations of the three laboratories for the same sample were extremely high( Figure 2 G).

[0170] Next, the present invention studied the influence of mapping and quantification algorithms on robustness. The standard SOP of the present invention uses the FANSe3 algorithm and the RPKM method to quantify the gene expression of the MHCC97H subculture dataset, reaching R 2 = 0.966 - 0.995( Figure 1 A). The present invention also tested two widely used RNA-seq algorithms, Bowtie2 and HISAT, and these algorithms showed lower R 2 = 0.963 - 0.991( Figure 2 H, I), indicating that the robustness of these algorithms is inferior to that of FANSe.

[0171] Finally, the present invention tested different mRNA enrichment strategies. The standard protocol of the present invention uses oligo-dT to enrich polyA+ mRNA (mature mRNA), which is applied in most studies. Another strategy is the rRNA depletion strategy (also known as ribosomal subtraction), which removes rRNA by probe hybridization or the RNaseH method. When the rRNA depletion strategy is adopted, the Pearson R 2 coefficient = 0.925 - 0.971( Figure 2 K, J), which is much lower than the polyA+ method.

[0172] Degrading the RNA of MHCC97H can achieve a relatively high transcriptome quantification stability

[0173] A good RNA reference standard should have relatively high robustness in actual use, and always obtain the same results even in the case of inevitable degradation. Because ribonucleases commonly present in the experimental environment can rapidly degrade ribonucleic acids. In addition, due to the possible different degrees of degradation of RNA standard samples during long-distance logistics transportation or storage, the standard samples may become inaccurate. In order to confirm whether the RNA of MHCC97H cells still has good stability after degradation, the present invention also tested such degraded RNA standard sample. First, the present invention created a scenario simulating degradation caused by environmental exposure: the RNA sample of MHCC97H was exposed to air for a long time, so the RNA enzymes in the environment might enter the test tube and degrade the RNA. This is the most common scenario in actual experiments. Electrophoresis showed that the total RNA of MHCC97H had different degrees of degradation(Figure 3 A). The present invention uses the oligo-dT method to enrich mRNA. Surprisingly, the SOP developed for non-degraded samples in the present invention can be directly used for degraded samples without any experimental and analytical changes, and highly comparable gene expression values can be generated even when using different alignment algorithms (Pearson R 2 > 0.91)( Figure 3 B, C, D).

[0174] MHCC97H can be used as a stable source cell line for the translatome standard

[0175] The present invention has found through transcriptome technology that MHCC97H cells have the highest stability among 5 different cell lines. To further illustrate the stability of MHCC97H at the proteomics level, the present invention detected the translatome samples of three cell lines, MHCC97H, HCCLM3, and HCCLM6, at consecutive passages, and found that the quantitative correlation of the MHCC97H translatome (i.e., quantification of RNC-mRNA) still reached above 0.93( Figure 4 ), while the correlations of HCCLM3 and HCCLM6 were only 0.91( Figure 5 ) and 0.82 (Figure 6) or above, indicating that MHCC97H has extremely high stability at the translatome level and is suitable as a source cell line for the translatome standard.

[0176] Evaluation of the quantitative stability of the MHCC97H proteome

[0177] Since the correlation between the translatome and the proteome is extremely high and proteins must be produced by the translation process, proteomics is the direct downstream of translatomics. The stability of the translatome can be reflected by detecting the proteome stability of MHCC97H. The present invention further detected the translatome and proteome of MHCC97H cells from passage 2 to passage 9 at the same time, used an Orbitrap Fusion Lumos mass spectrometer to identify peptide fragments, and formed 9 DDA and 9 DIA data sets. By using the Target-decoy library search strategy to reduce the false positive rate of identification, the qualitative and quantitative lists of the translatome and proteome of cells between different passages were finally obtained. The correlation R of the translatome of MHCC97H cells from passage 2 to passage 9 2 = 0.948 - 0.991( Figure 7 A), and a correlation analysis was performed on the protein quantitative results. The quantitative correlation between different passages of proteins in the DDA mode reached R 2 = 0.934 - 0.976( Figure 7 B), and the quantitative correlation between different passages of proteins in the DIA mode reached R = 0.942 - 0.986( Figure 7C); In the present invention, the 3rd passage of MHCC97H cells were used for three independent enzymatic digestion experiments, and the quantitative correlation was 0.945 - 0.949 in DDA mode ( Figure 7 D), and the quantitative correlation was 0.975 - 0.99 in DIA mode ( Figure 7 E). The nucleic acid and protein quantitative correlations among different passages of the candidate reference standard MHCC97H reached an extremely high level, showing high stability at both the nucleic acid and protein levels, further indicating that the MHCC97H cell line can continuously and stably produce translational group reference samples.

[0178] Mass spectrometry consistency evaluation of MHCC97H proteome samples by third-party laboratories

[0179] Good protein reference standards should achieve similar identification and quantification effects in different laboratories and have high stability. In the present invention, 1 mg of the whole protein of the same batch of reference standard MHCC97H cell line samples was given to each of South China University of Technology (SCUT), Beijing Normal University (BNU), Dalian Institute of Chemical Physics, Chinese Academy of Sciences (DICP), and Jinan University (JNU). All laboratories adopted standardized enzymatic digestion and mass spectrometry experimental procedures. The quality control methods were as follows: The standard operation of enzymatic digestion with an ultrafiltration tube was adopted for the experiment. After enzymatic digestion, the peptides were desalted and the peptide concentration was detected. After enrichment, the peptide concentration was ≥1 μg / μL. The instruments of three laboratories were all unified as Thermo Orbitrap Fusion Lumos, and one used Q Exactiveplus ( Figure 8 A). The false positive rate of identification was reduced by the target-decoy library search strategy, and finally the protein qualitative and quantitative lists of the same sample in different laboratories were obtained. The pairwise KS statistical analysis was performed on the isoelectric point distributions of the proteins identified by different laboratories, and no statistically significant differences were obtained (P value > 0.05) ( Figure 8 B), indicating that the isoelectric point distributions of the proteins identified from the same sample were relatively consistent under the tests of the four laboratories. The DDA iBAQ quantitative consistency test was performed on the protein quantitative results of the four laboratories. The quantitative repeatability R of the same laboratory 2 = 0.925 - 0.949 ( Figure 8 C), and the DIA quantitative correlation R between different laboratories 2 = between 0.832 - 0.926 ( Figure 8 D). It should be noted that because the mass spectrometry instrument model used by SCUT is different from the other three, the correlation is lower compared with the other three experiments. Under the mass spectrometry identification of different laboratories, the protein mass spectrometry quantification of the MHCC97H cell line still achieved extremely high repeatability and verifiability.

[0180] Conclusion

[0181] By continuously collecting 5 cell lines such as MHCC97H for multiple generations and performing transcriptome sequencing, it is concluded that among the 5 experimental cell lines, the quantitative correlation of the transcriptome of the 5th - 14th generation cells of MHCC97H is the highest, with R 2 reaching above 0.97, and the conclusion that the transcriptome is the most stable during the passage of MHCC97H cells is obtained. Further evaluating the stability of the translatome of the MHCC97H cell line, it is found that the quantitative correlation R of RNC - seq of the MHCC97H cell line reaches above 0.9, and the quantitative result R of protein mass spectrometry also reaches above 0.92. Moreover, repeatable and verifiable stable data are also obtained under the operation of a third - party laboratory according to the standard process. This result shows that MHCC97H is very stable and can be used as a reference material for long - term production and can be widely promoted and used in various laboratories.

[0182] In addition, the results obtained from the research of the present invention are based on 5 cell lines, namely MHCC97H, HCCLM3, HCCLM6, A549, and Hela. It can only be said that under the unified standard operation process, the stability of the reference standard MHCC97H selected from these 5 experimental strains is the strongest. The reason why the MHCC97H cell line is more stable than other experimental cell lines may be that during the continuous culture of cells for multiple generations, compared with other experimental strains, the growth rate of the MHCC97H cell line is relatively slow and the basal metabolic rate is relatively low, which may be the reason for its high stability.

[0183] In the 5 cancer cell lines, through the evaluation of the pass - through consistency test of the MHCC97H cell line in terms of qualitative and quantitative accuracy, it is concluded that the stability of the MHCC97H cell line is extremely high and it can be stably produced as a reference standard for omics data output. Moreover, the optimized unified sequencing process and mass spectrometry process of the present invention also have extremely high robustness and can be widely transplanted and promoted in various laboratories. In short, the research of the present invention aims to develop the key technologies and application demonstrations for the quality control of each life omics data, establish the standard methods and automated tools for the whole - process quality control of life omics data, promote the application of life omics data in basic medicine and clinical medicine, accelerate the effective conversion and practical application of life omics data in the field of precision medicine, and benefit patients with various diseases. At the same time, further enhance the international influence of China's life omics technology and make precision medicine an important part of the national strategic emerging industries.

[0184] All documents mentioned in this invention are cited herein by reference as if each individual document was cited by reference. In addition, it should be understood that after reading the above teachings of this invention, those skilled in the art can make various changes or modifications to this invention, and these equivalent forms also fall within the scope defined by the appended claims of this application.

Claims

1. Use of total RNC-mRNA extracted from the MHCC97H cell line in the preparation of a translatome standard.

2. The application according to claim 1, characterized in that: The total RNC-mRNA is prepared by the following method: Step 1, culture MHCC97H cells; Step 2, isolate RNC from the cells; Step 3, isolate RNC-mRNA from the RNC.

3. The application according to claim 2, wherein: The specific operation of Step 2 is as follows:

1. When the cells reach confluence in a 75 cm 2 culture flask, add cycloheximide to a final concentration of 100 μg / mL and incubate in an incubator for 15 min; 2. Discard the culture supernatant, place the culture flask on ice, and wash twice with pre-cooled PBS.

3. Discard all PBS completely, add 2 mL of pre-cooled RB lysis buffer, and lyse on ice for 30 min.

4. Scrape the cells with a cell scraper and transfer the lysate to a pre-cooled EP tube.

5. Centrifuge at 13200 rpm at 4 °C for 10 min.

6. Add 15 mL of pre-cooled 30% sucrose solution to an ultracentrifuge tube.

7. Transfer the supernatant from Step 5 to the ultracentrifuge tube.

8. Ultracentrifuge at 42500 rpm at 4 °C for 5 h.

9. Discard the supernatant, carefully wash the surface of the RNC pellet twice with pre-cooled sterile PBS; obtain RNC.

4. The application according to claim 2, characterized in that: The specific operation of Step 3 is as follows:

1. Add 1 mL of Trizol to the RNC pellet obtained in Step 2. Add 0.2 mL of chloroform to every 1000 microliters of Trizol, shake vigorously for 15 s, and let stand at room temperature for 3 min.

2. Centrifuge at 12000 ×g at 4 °C for 15 min to separate the sample into three layers.

3. Pipette the upper aqueous phase and transfer it to a new 1.5 mL EP tube. Do not pipette the middle layer liquid during the operation.

4. Add 800 µL of isopropanol and invert to mix well.

5. Place at -20 °C and let stand for 8 h.

6. After 8 h, centrifuge at 12000 ×g at 4 °C for 30 min to remove the supernatant.

7. Add 1 mL of 75% ethanol; the ethanol needs to be pre-cooled at -20 °C for 30 min in advance.

8. Centrifuge at 7500 ×g at 4 °C for 5 min to remove the supernatant.

9. Repeat Steps 7 and 8 once.

10. Remove the residual ethanol, open the tube cap, and air dry for 5 min.

11. Dissolve according to the particle size by adding 30 - 60 microliters of RNase Free pure water. This step obtains the RNC-mRNA standard, which is stored in an -80 °C refrigerator.

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