A detection method and kit based on a needle puncture sample

Abstract

The application discloses a detection method and a kit based on a one-needle puncture sample. The detection method based on the one-needle puncture sample comprises the following steps: performing single-nucleus extraction treatment on all the one-needle puncture samples to obtain single-nucleus samples; taking a part of the single-nucleus samples for single-nucleus acid detection; directly using the remaining single-nucleus samples for whole-transcriptome detection and / or whole-genome detection; or, freezing the remaining single-nucleus samples for the whole-transcriptome detection and / or the whole-genome detection. The detection method based on the one-needle puncture sample can realize the detection of two omics simultaneously on the same one-needle puncture tissue, maximizes the utilization rate of the puncture tissue which is a kind of biological sample with a very small sample amount; moreover, the detection method avoids the increase of patient trauma caused by multiple puncture sampling, and avoids the serious heterogeneity problem of two omics data, thereby reducing the difficulty of later multi-omics analysis.

Description

Technical Field

[0001] This application relates to the field of puncture sample detection technology, and in particular to a detection method and kit based on a single-needle puncture specimen. Background Technology

[0002] Most human RNAs are associated with cancer. Overexpression or suppression of tumor suppressor RNAs can lead to tumor growth by inhibiting apoptosis, cell proliferation, epithelial-mesenchymal transition (EMT), and metastasis. With the widespread adoption and rapid decrease in cost of high-throughput gene sequencing technology, transcriptome sequencing (RNA-seq) is increasingly used in cancer research. RNA-seq uses high-throughput gene sequencing (HTS) technology to determine almost all transcript sequences in a specific tissue under a given condition, and is one form of genome sequencing. RNA-seq data contains rich information on gene fusions, gene expression variations, transcript variations, gene sequence variations, and immunogene diversity. In recent years, the application of RNA-seq has increased, improving our ability to diagnose subtyping, prognostic stratification, and personalized treatment. Some centers have begun to apply RNA-seq to clinical case analysis and have demonstrated its value.

[0003] Single-cell RNA sequencing (snRNA-seq) is a technique that extracts single-cell nuclei from a sample, isolates and labels the nuclei, and studies nuclear gene expression at the single-cell level. It overcomes the limitations of bulk RNA sequencing (BRNA-seq) in obtaining information about cellular heterogeneity. Clinical tissues are mixtures of various cells; for example, tumor cells at the center, periphery, and distant metastases differ in cell type and transcriptome. Traditional bulk RNA-seq extracts mixed RNA from tissues or a group of cells for sequencing, obtaining the average gene expression of all cells, which masks the expression specificity of individual cells. SnRNA-seq, however, effectively addresses the issue of cellular heterogeneity. By detecting the expression profile of individual cells, it identifies cell populations and enables in-depth research into tumor metastasis, intratumoral heterogeneity, microenvironment reprogramming, and drug resistance. In recent years, it has been widely applied in biological research fields such as oncology, immunology, growth, and development.

[0004] A needle biopsy (also known as a puncture biopsy) is a surgical procedure that obtains cellular samples from a patient's body for laboratory testing. Common needle biopsy techniques include fine-needle aspiration and core needle biopsy. Needle biopsies can be used to collect tissue or fluid samples from muscles, bones, and other organs such as the liver or lungs. Both puncture and surgical sampling can yield pathological specimens. However, puncture sampling is less invasive and allows for multi-site sampling, making it crucial for the clinical diagnosis and scientific research of tumors. Puncture sampling can not only determine the tissue classification but also whether it is benign or malignant, and it can also provide a basis for subsequent treatment.

[0005] The sample obtained by puncture sampling is called a puncture specimen. Since the sample size of the puncture specimen is very small, multiple puncture samplings are usually required when performing different nucleic acid tests. This not only increases the trauma to the patient, but also increases the difficulty of subsequent multi-omics analysis due to the heterogeneity of different punctures.

[0006] Therefore, how to address the increased patient trauma and heterogeneity caused by multiple punctures remains a key research focus in the field of puncture biopsy technology. Summary of the Invention

[0007] The purpose of this application is to provide an improved detection method and kit based on a single-needle puncture specimen.

[0008] To achieve the above objectives, this application adopts the following technical solution:

[0009] One aspect of this application discloses a detection method based on single-needle aspiration specimens, comprising: extracting single cell nuclei from all single-needle aspiration specimens to obtain single-cell nucleus samples; using a portion of the single-cell nucleus samples for single-cell nucleic acid detection, and directly using the remaining single-cell nucleus samples for whole transcriptome and / or whole genome detection; or, cryopreserving the remaining single-cell nucleus samples for future use in whole transcriptome and / or whole genome detection. Cryopreservation can be performed using conventional methods, such as freezing at -80°C.

[0010] It should be noted that in practical testing, due to the small sample size of aspiration specimens, it is not possible to directly use a portion of the specimen for single-cell nucleic acid detection and another portion for whole transcriptome and / or whole genome detection; the losses during the extraction process would prevent both omics experiments from succeeding. Furthermore, even if single-cell nucleus extraction is performed separately from the two portions of the specimen, it is still impossible to achieve both omics detections; this is because the aspiration tissue contains other residues besides single-cell nuclei. Directly dividing the two portions of the aspiration tissue for separate single-cell nucleus extraction results in a highly uncertain number of single-cell nuclei that can be extracted from each portion, and the losses from separate single-cell nucleus extraction are even greater, thus failing to guarantee the success of both omics experiments. Therefore, this application creatively proposes to first perform single-cell nucleus extraction on all single-needle aspiration specimens to obtain single-cell nucleus samples; then, at the single-cell level, a portion is extracted for single-cell nucleic acid detection, and the remainder is used for whole transcriptome and / or whole genome detection. This effectively ensures the success of both omics experiments. Furthermore, the detection method of this application completes two omics tests with a single puncture, eliminating the need for multiple punctures and sampling. This avoids increased patient trauma caused by multiple punctures and sampling, as well as the serious heterogeneity problem between the two omics data, thereby reducing the difficulty of subsequent multi-omics analysis.

[0011] It should also be noted that the puncture specimen detection method of this application mainly achieves nucleic acid detection of two omics through a single puncture specimen. The detection result only obtains the genetic information of the sample, and does not directly obtain the diagnosis result of the disease. Therefore, the puncture specimen detection method of this application does not belong to the diagnosis and treatment method of disease.

[0012] In one implementation of this application, single-cell nucleic acid detection is single-cell nuclear transcriptome sequencing detection and / or single-cell whole genome sequencing detection.

[0013] It should be noted that the key to this application is to achieve the detection of two omics based on the same needle puncture tissue. As for the specific single-cell nucleic acid detection, existing technologies can be used to perform single-cell nuclear transcriptome sequencing and / or single-cell whole genome sequencing.

[0014] In one implementation of this application, a single-needle puncture specimen is obtained using a sampling needle with a specification of 16g / 20mm or larger. The 16g / 20mm sampling needle is merely one specific sampling needle used in this implementation; the detection method of this application may also be applied to smaller or larger sampling needles. It is understood that, under the same conditions, a larger sampling needle can obtain more samples, and therefore, the detection method of this application can also be used to perform two-omics detection. As for smaller sampling needles, if a sufficient number of single-cell nucleus samples can be obtained to meet the requirements of two-omics detection, the detection method of this application can also be used.

[0015] In one implementation of this application, the single-cell nucleus extraction process includes obtaining a single-cell nucleus sample by grinding and separating the puncture tissue obtained from a single needle puncture.

[0016] In one implementation of this application, obtaining a single-cell nucleus sample by grinding and separation includes: cutting the punctured tissue into small pieces in a homogenization buffer; grinding the tissue pieces using a tissue grinder and filtering them using a cell sieve to remove residue and collect the filtrate; centrifuging the filtrate for the first time and discarding the supernatant; resuspending the precipitate with nuclear washing buffer, centrifuging for the second time, discarding the supernatant, and repeating this step at least once; and resuspending the precipitate with nuclear buffer to obtain the single-cell nucleus sample.

[0017] In one implementation of this application, obtaining a single-cell nucleus sample through grinding and separation specifically includes the following steps:

[0018] 1) Place the tissue homogenizer on ice and add pre-cooled homogenization buffer (HB);

[0019] 2) Place the aspirated tissue in a culture dish containing pre-cooled homogenization buffer;

[0020] 3) Cut the aspirated tissue into small pieces and transfer the tissue pieces and homogenization buffer together to a pre-cooled tissue homogenizer;

[0021] 4) Let stand until the tissue block is soaked, then grind the tissue block vertically with a grinding rod until the resistance decreases to obtain a tissue homogenate.

[0022] 5) Filter the tissue homogenate using a cell sieve and collect the filtrate into a pre-cooled centrifuge tube; at the same time, wash the tissue homogenizer and cell sieve with homogenate buffer and collect the washing solution into the same centrifuge tube;

[0023] 6) Rinse the inside of the tissue homogenizer with nuclease-free water (NF water) to remove residual tissue and discard the washing solution; transfer the tissue homogenate in the centrifuge tube to the rinsed tissue homogenizer for a second homogenization.

[0024] 7) Filter the tissue homogenate after the second grinding using a cell sieve, and collect the filtrate into a new centrifuge tube;

[0025] 8) Wash the tissue homogenizer and cell strainer with homogenization buffer and collect the washing solution into the same centrifuge tube;

[0026] 9) Centrifuge the filtrate collected in step 8) and discard the supernatant;

[0027] 10) Resuspend the precipitate in nuclei wash buffer, then centrifuge and discard the supernatant;

[0028] 11) Repeat step 10) at least once;

[0029] 12) The precipitate was resuspended in nuclear buffer to obtain a nuclear suspension;

[0030] 13) Single-cell samples were obtained by isolating from the cell nucleus suspension.

[0031] In one implementation of this application, cell sieve filtration includes: firstly, using a 70μm to 90μm cell sieve for a first filtration, collecting the filtrate and grinding it again, then using a 30μm to 40μm cell sieve for a second filtration, collecting the filtrate and centrifuging it for a first time.

[0032] In one implementation of this application, the conditions for the first centrifugation are: 4°C, 500g, centrifugation for 5-8 minutes.

[0033] In one implementation of this application, the conditions for the second centrifugation are: 4°C, 800g, for 8-10 minutes. The centrifugation temperature of 4°C is a conventional low-temperature centrifugation temperature in the art. Based on this, as long as the low-temperature conditions can be ensured, experimentally permissible adjustments to this temperature are possible. As for the centrifugation speeds for both centrifugations, the aim is to effectively precipitate cell nuclei and remove unwanted impurities; similarly, experimentally permissible adjustments can be made based on the centrifugation speeds specified in this application.

[0034] In one implementation of this application, the homogenization buffer comprises 1× basal buffer, 240 mmol / L–260 mmol / L sucrose, 0.9%–1.1% BSA, 0.9 mmol / L–1.1 mmol / L DTT, 1× protease inhibitor cocktail, and 0.3 U / μL–0.5 U / μL ribonuclease inhibitor; wherein the 1× basal buffer comprises 18 mmol / L–22 mmol / L Tris, 22.5 mmol / L–27.5 mmol / L KCl, and 4.5 mmol / L–5.5 mmol / L MgCl2. The 1× basal buffer can be adjusted appropriately according to experimental requirements; for example, a 0.9–1.1× basal buffer can also be used. Similarly, the 1× protease inhibitor cocktail can be adjusted from 0.9–1.1× protease inhibitor cocktail as needed.

[0035] In one implementation of this application, the cell nucleus washing solution is a homogenization buffer containing iodixanol.

[0036] In one implementation of this application, the concentration of iodixanol in the cell nucleus washing solution is 20% to 25%.

[0037] In one implementation of this application, the nuclear buffer is a PBS buffer containing 0.9% to 1.1% BSA, and also contains 0.4 U / μL to 0.5 U / μL of ribonuclease inhibitor.

[0038] Another aspect of this application discloses a kit for the detection method of this application, comprising a 1 mol / L sucrose solution, 6× basal buffer, BSA, 100 mmol / L DTT, a protease inhibitor mixture, a 40 U / μL ribonuclease inhibitor, 60% iodixanol, PBS buffer, and nuclease-free water; wherein, the 1 mol / L sucrose solution is obtained by dissolving sucrose in water; the solvent of the 6× basal buffer is nuclease-free water, which contains 120 mmol / L Tris, 150 mmol / L KCl, and 30 mmol / L MgCl2; one tablet of the protease inhibitor mixture is dissolved in 0.5 mL of nuclease-free water to obtain a 100× protease inhibitor mixture. In this kit, the concentrations of each component are primarily for ease of storage and subsequent use. Based on these, appropriate adjustments can be made according to requirements. For example, the concentration of sucrose solution can be 0.9 mol / L to 1.1 mol / L, the basal buffer can be 5.9 × to 6.1 ×, the concentration of DTT can be 90 mmol / L to 100 mmol / L, the concentration of ribonuclease inhibitor can be 39 U / μL to 41 U / μL, and the concentration of iodixanol can be 50% to 61%.

[0039] It should be noted that the kit of this application mainly contains reagents for obtaining single cell nuclei by grinding and separating; it is understood that, for ease of use, it may also contain reagents for single-cell nucleic acid detection, as well as reagents for whole transcriptome detection and / or whole genome detection; of course, reagents for single-cell nucleic acid detection, whole transcriptome detection, and whole genome detection can also be obtained by purchasing commercially available products, and no specific limitation is made here.

[0040] In one implementation of this application, the kit is prepared using a homogenization buffer consisting of 6× basal buffer, 1 mol / L sucrose solution, 10% BSA aqueous solution, 100 mmol / L DTT, 100× protease inhibitor mixture, 40 U / μL ribonuclease inhibitor, and nuclease-free water. The homogenization buffer is generally prepared fresh for each use.

[0041] In one implementation of this application, the kit is prepared by using 60% iodixanol and 6× basal buffer to prepare a 50% iodixanol solution, and then using the 50% iodixanol solution and homogenization buffer to prepare a cell nucleus washing buffer. The cell nucleus washing buffer is generally prepared fresh for use, but can also be stored at 4°C for 30 days.

[0042] In one implementation of this application, the reagent kit is used to prepare a nuclear buffer using PBS buffer containing 10% BSA, 40 U / μL ribonuclease inhibitor, and PBS buffer. The nuclear buffer is generally prepared fresh for use.

[0043] The beneficial effects of this application are as follows:

[0044] This application is based on a single-needle puncture specimen detection method, which enables the simultaneous detection of two omics in the same puncture tissue, maximizing the utilization of biological specimens with small sample sizes such as puncture tissue; moreover, it avoids increased patient trauma caused by multiple puncture sampling and also avoids the serious heterogeneity problem of the two omics data, thereby reducing the difficulty of subsequent multi-omics analysis. Attached Figure Description

[0045] Figure 1 This refers to the detection results of the whole transcriptome library of Sample 1 in the embodiments of this application;

[0046] Figure 2 This refers to the detection results of the whole transcriptome library of Sample 2 in the embodiments of this application;

[0047] Figure 3 This refers to the detection results of the whole transcriptome library of Sample 3 in the embodiments of this application;

[0048] Figure 4This refers to the detection results of the whole transcriptome library of Sample 4 in the embodiments of this application;

[0049] Figure 5 This refers to the detection results of the whole transcriptome library of sample 5 in the embodiments of this application;

[0050] Figure 6 This is the sequencing data analysis result of sample 1 in the embodiments of this application;

[0051] Figure 7 This is another analysis result of the sequencing data of sample 1 in the embodiments of this application;

[0052] Figure 8 This is a statistical result of the number of sequencing reads of nuclear RNAseq from five samples in the embodiments of this application;

[0053] Figure 9 This is a statistical result of the proportion of rRNA reads from the nuclear RNAseq of five samples in the embodiments of this application;

[0054] Figure 10 This is a statistical result of the proportion of clean reads from the nuclear RNAseq of five samples in the embodiments of this application;

[0055] Figure 11 This is a statistical result showing the proportion of uniquely aligned reads from the nuclear RNAseq of the five samples in this application embodiment;

[0056] Figure 12 This is a statistical result of the number of genes with TPM>1 detected by nuclear RNAseq of five samples in the embodiments of this application;

[0057] Figure 13 This is a statistical result of the number of gene fusions detected by nuclear RNAseq of five samples in the embodiments of this application. Detailed Implementation

[0058] Existing puncture-based tissue library construction and sequencing methods cannot achieve both single-cell nuclear transcriptome sequencing and whole transcriptome detection with a single puncture, meaning it is impossible to complete two omics tests with a single puncture. While using multiple puncture specimens can perform single-cell nuclear transcriptome sequencing and whole transcriptome sequencing separately, it not only increases patient trauma but also complicates subsequent multi-omics analysis due to the heterogeneity of different puncture sites.

[0059] This application argues that the utilization rate of biological specimens with small sample sizes, such as aspirated tissue, has not been maximized. Therefore, this application creatively develops a single-cell nuclear transcriptome and whole transcriptome application based on a single-needle aspiration specimen, enabling both single-cell nuclear transcriptome sequencing and whole transcriptome detection, achieving two omics applications from a single needle aspiration.

[0060] This application is based on a detection method for a single-needle aspiration specimen, which includes isolating a single-cell sample from the single-needle aspiration specimen; dividing the single-cell sample into at least two parts, one part for single-cell nucleic acid detection and the other part for whole transcriptome detection and / or whole genome detection; thereby enabling the detection of at least two omics in a single-needle aspiration specimen.

[0061] The main process of this technology includes: after isolating a single-cell sample from a single-needle aspiration specimen, a portion of the cell nucleus is isolated for single-cell nucleic acid detection, while the remaining cell nucleus is used for whole transcriptome and / or whole genome detection. For example, the remaining cell nucleus can be washed with RNase offH2O after centrifugation, and then RNA can be extracted directly; or it can be washed with RNase offH2O after centrifugation, then 250 μL of RNase offH2O + 750 μL of trizol LS is added and stored at -80°C. When needed, RNA is extracted from the cell nucleus using the trizol method for RNA library construction and sequencing. If the remaining cell nucleus is for whole genome detection, DNA is extracted from the cell nucleus for genomic analysis.

[0062] The detection method of this application is based on simultaneously performing single-cell nuclear transcriptome sequencing and whole-nuclear transcriptome sequencing on the same needle-punctured tissue, enabling the sequencing of punctured tissue (e.g., with a 16g / 20mm sampling needle, the punctured tissue volume is approximately 1mm²). 2 *15mm) This maximizes the utilization of biological specimens with small sample sizes. It reduces the heterogeneity of performing two omics analyses separately using multi-needle puncture specimens (during tumor growth, after multiple divisions and proliferations, the daughter cells exhibit molecular biological or genetic changes, resulting in differences in tumor growth rate, invasiveness, drug sensitivity, prognosis, etc.), and reduces the difficulty of subsequent multi-omics analysis.

[0063] The present application will be further described in detail below through specific embodiments. The following embodiments are only for further illustration of the present application and should not be construed as limiting the present application.

[0064] Unless otherwise specified, the technical means used in the embodiments are conventional means well known to those skilled in the art.

[0065] Example

[0066] I. Materials and Methods

[0067] 1. Single cell nucleus extraction

[0068] 1.1 Experimental Preparation

[0069] Clean bench preparation: Turn on the clean bench fan, first wipe the entire surface with 75% alcohol, then use DNA-OFF (a DNA remover) to thoroughly wipe the inside of the clean bench and instruments, especially the surfaces of metal and plastic products. Turn off the fan and sterilize by UV irradiation for 30 minutes. Before processing tissues, wipe the inside of the clean bench thoroughly with RNase-ZAP using the same method. After wiping, experiments can begin directly.

[0070] Cleaning and sterilization of tissue homogenizers and surgical instruments: Clean the entire homogenizer set twice with ddH2O to remove visible impurities; transfer it to a laminar flow hood and clean it once with 75% alcohol; wipe it once with DNA-OFF and clean it twice with ddH2O; after drying, irradiate the homogenizer with UV light for 30 minutes; wipe it once with RNase-Zap and rinse it at least three times with DEPC water until the foam is completely rinsed away.

[0071] Centrifuge parameters (low-temperature horizontal centrifuge): temperature 4℃, speed 500g, centrifugation time 5 minutes. Before centrifugation, plug in the centrifuge power supply 10 minutes in advance and start the centrifuge to ensure it reaches a low operating temperature of 4℃. Maintain low temperature throughout the entire operation, such as operating in an ice bath.

[0072] 1.2 Reagent Preparation

[0073] 1) 1 mol / L sucrose solution (stored at 4℃ for 30 days)

[0074] reagents Final concentration Prepare 50mL of solution sucrose 1M 17.12g <![CDATA[H2O]]> / 39.25mL

[0075] After the sucrose is completely dissolved, filter it through a 0.2 μm filter into a new 50 mL centrifuge tube, store at 4 °C, and use within 30 days.

[0076] 2) 6× Basic Buffer

[0077] reagents Final concentration Prepare 10mL of the solution. 1 mol / L Tris, pH 7.8 120mmol / L 1.2mL 1mol / LKCl 150mmol / L 1.5mL <![CDATA[1mol / LMgCl2]]> 30mmol / L 0.3mL Nuclease-freewater / 7.0mL

[0078] Store at 4℃ and use within 30 days.

[0079] 3) Homogenization Buffer (HB)

[0080] reagents Final concentration Prepare 10mL of the solution. Prepare a 6mL dosage 6×BasicBuffer 1× 1.67mL 1.00mL 1 mol / L sucrose solution 250mmol / L 2.50mL 1.50mL <![CDATA[10%BSAinH2O]]> 1％ 1.00mL 0.60mL 100 mmol / L DTT 1mmol / L 0.10mL 0.06mL 100×Protease inhibitor cocktail 1× 0.10mL 0.06mL RNase inhibitor (40 U / μL) 0.4 U / μL 0.10mL 0.06mL Nuclease-freewater / 4.53mL 2.72mL

[0081] Prepare fresh for each use. 6 ml for 2 ml Dounce, 10 ml for 7 ml Dounce per tissue sample; 100×Protease inhibitor cocktail preparation: dissolve 1 cocktail tablet in 0.5 mL of NF water.

[0082] 4) 50% Iodixanol Solution

[0083] reagents Final concentration Prepare 12mL of solution Optiprep (60% iodixanol) 50％ 10.0mL 6×BasicBuffer 1× 2mL

[0084] Store at 4℃ and use within 30 days. Use 2 mL per tissue sample.

[0085] 5) Nuclei Resuspension Buffer (NRB)

[0086] reagents Final concentration Prepare a 2mL dosage 10% BSA in PBS 1％ 0.2mL RNase inhibitor (40 U / μL) 0.4 U / μL 0.02mL 1×PBS / 1.78mL

[0087] Prepare fresh for use, 3 mL per tissue sample.

[0088] 1.3 Obtaining tissue homogenate and nucleus (2 mL tissue homogenizer)

[0089] 1) Place the tissue homogenizer on ice and add 0.9 mL of pre-chilled Homogenization Buffer.

[0090] 2) Remove the punctured tissue from the liquid nitrogen and immediately place it in a 30 mm culture dish containing 0.9 mL of pre-cooled Homogenization Buffer.

[0091] 3) Use surgical scissors to cut the punctured tissue into 1mm pieces. 3 Small pieces are transferred together with the liquid using a wide-mouth pipette tip to a pre-cooled tissue homogenizer.

[0092] 4) Let stand for 2 minutes to allow the tissue to fully infiltrate; use abrasive rod A to grind the tissue block vertically until the resistance is significantly reduced, approximately 10 times.

[0093] 5) Filter the tissue homogenate using a 70 μm cell sieve and collect the tissue homogenate into a pre-cooled 50 mL centrifuge tube; wash the homogenizer and cell sieve with 0.5 mL of Homogenization Buffer and collect the washing solution into the same 50 mL centrifuge tube.

[0094] 6) Clean the inside of the grinder with 3 mL of NF-H2O to remove residual tissue and discard as much of the cleaning liquid as possible; transfer the tissue homogenate to the cleaned grinder and grind it 5 times with grinding rod B.

[0095] 7) Filter the tissue homogenate after the second grinding into a 15mL centrifuge tube using a 30μm cell sieve.

[0096] 8) Wash the grinder and cell sieve with 0.5 mL of Homogenization Buffer and collect the washing solution into the same 15 mL centrifuge tube.

[0097] 9) Centrifuge the filtrate collected in step 8) at 4°C and 500g for 5 minutes, and discard the supernatant.

[0098] Meanwhile, depending on the sample type, 5 mL of 20% Nuclei Wash Buffer was prepared using Homogenization Buffer and 50% Iodixanol Solution.

[0099]

[0100] Wherein, “%iodixanol(w / v)” refers to the mass-volume percentage of iodixanol in the final solution, “η” refers to the dynamic viscosity of the liquid, “ρ” refers to the density of the liquid, “50%iodixanol” refers to the volume of 50% iodixanol solution to be added, and “HB” refers to the volume of HB to be added.

[0101] 10) Add 2.4 mL of Nuclei Wash Buffer to the precipitate to resuspend it, and gently pipette to mix. At this point, the iodixanol concentration is 20%. Then, centrifuge at 800 g for 10 minutes at 4 °C and discard the supernatant.

[0102] Note: The upper layer contains impurities at this point. Slowly remove it and retain the white precipitate at the bottom.

[0103] 11) Repeat step 10), that is, add 2.4 mL of Nuclei Wash Buffer to the white precipitate at the bottom to resuspend the precipitate, gently pipette to mix, and then centrifuge at 800 g for 10 minutes at 4 °C and discard the supernatant.

[0104] 12) Depending on the amount of precipitate, resuspend the cell nuclei in 0.5-1.5 mL of Nuclei Resuspension Buffe and mix by pipetting.

[0105] 13) Nucleus counting: Based on the nucleus density and the required number of nuclei, calculate the required volume of the original nucleus solution and the volume of the cell resuspension solution. The nucleus density is counted using a hemocytometer.

[0106] Before loading the chip, aspirate the corresponding volume of cell nuclear suspension into a 1.5 mL centrifuge tube, centrifuge at 500 g for 5 minutes at 4 °C, and discard the supernatant; then, prepare the cell nuclear suspension according to the calculated amount, gently pipette to mix, and perform chip experiments and cDNA and oligo library construction.

[0107] This experiment tested 5 prostate tissue puncture samples. Each sample was a single-needle puncture sample. All puncture samples were tested using the BARD fully automated biopsy puncture gun (MG15-22) and the matching BARD biopsy gun puncture needle (16G, outer diameter of 1.6mm, puncture length of 1.5cm).

[0108] 2. Single-cell nuclear transcription library

[0109] The extraction of RNA from the single-cell nuclear transcription library preparation kit was performed according to the MGI website DNBelab C series high-throughput single-cell RNA library preparation kit V2.0 (No.: H-020-000553-00).

[0110] After the single-cell nuclear transcription library was assembled, DNBSEQ sequencing was used for detection.

[0111] 3. Cell nucleus preservation and RNA extraction

[0112] 3.1 Preservation of cell nuclei

[0113] 1) After single-cell extraction, the remaining cell nuclei are washed with RNase offH2O after centrifugation and then used directly for RNA extraction using the QIAamp DNA Mini Kit. Alternatively, after centrifugation, the nuclei are washed with 1 mL of RNase offH2O, centrifuged at 800g for 10 minutes at 4°C, and the supernatant is discarded; then 250 μL of RNase offH2O and 750 μL of trizol LS are added, the mixture is inverted and mixed, and stored at -80°C for about one month.

[0114] 2) Take 900 μL of cell nuclei stored in trizol LS, thaw them, add 250 μL of chloroform, vortex to mix, centrifuge at 12000 rpm, 4℃ for 15 min, remove the tube and try not to shake the liquid in the tube to keep it in layers.

[0115] 3) Transfer the colorless liquid from the top layer to a new 1.5 mL EP tube, add 1.2 μL of glycogen and 600 μL of isopropanol, mix by inverting, and incubate at -20°C for at least 2 hours.

[0116] 4) Take out the mixture that has been placed at -20℃ for 2 hours, centrifuge at 12000 rpm and 4℃ for 15 minutes, discard the supernatant, and avoid aspirating the precipitate when using a pipette.

[0117] 5) Wash the precipitate with 75% ethanol, centrifuge at 12000 rpm and 4℃ for 5 min, and discard the supernatant.

[0118] 6) Repeat the washing with 75% ethanol, that is, add 75% ethanol to the precipitate and wash again, centrifuge at 12000 rpm, 4℃ for 5 min, carefully remove the residual liquid with a pipette, and air dry at room temperature for 10 min; then dissolve in 20 μL DEPC and store at -80℃.

[0119] 3.2 Extraction and library construction of nuclear RNA

[0120] In this experiment, RNA was extracted from five samples using different methods, and libraries were constructed. Specifically, Samples 1 and 2 were cell nuclei remaining after single-cell extraction. After centrifugation, they were washed with RNase offH2O, and RNA was extracted directly using the QIAamp DNAMiniKit kit.

[0121] Samples 3 to 5 were cell nuclei remaining after single-cell extraction. They were preserved in Trizol LS and RNA was extracted using the Trizol method.

[0122] The extracted RNA was detected using Qsep400.

[0123] The extraction of nuclear transcriptome library was performed using the MGI website's MGI Easy rRNA Removal Kit V1.3 (catalog number: 940-001751-00) and MGI Easy RNA Library Preparation Kit (catalog number: 1000006383).

[0124] RNA libraries were analyzed using the ssDNAqubit quantification method or equipment.

[0125] The constructed whole transcriptome RNA library was sequenced using DNBSEQ.

[0126] II. Data Results

[0127] 1. Results of single-cell nucleus extraction and isolation

[0128] This experiment tested a total of 5 samples, each of which was a single-needle puncture sample. The results of single-nucleus extraction are shown in Table 1. 75,000 nuclei from each sample were used to construct a single-nucleus transcription library, and the remaining nuclei were used for nuclear RNA extraction.

[0129] Table 1 Results of single cell nucleus extraction

[0130] sample Number of nuclei examined under a microscope (number of nuclei) Nucleus concentration (cups / μL) Nuclear fluid (μL) Kernel removal Total number of cores Remaining cores (number) Sample1 64 800 1000 93.8 800000 725000 Sample2 101 1262.5 300 59.4 378750 303750 Sample3 106 1325 500 56.6 662500 587500 Sample4 89 1112.5 500 67.4 556250 481250 Sample5 84 840 500 89.3 420000 345000

[0131] In Table 1, "Nucleus count observed under a microscope" refers to the number of cell nuclei observed using a microscope; "Nucleus concentration" refers to the concentration of the Nuclei Resuspension Buffe resuspended cell nucleus suspension obtained by dividing "Nucleus count observed under a microscope" by the volume observed under a microscope; "Nucleus solution" refers to the total volume of the prepared Nuclei Resuspension Buffe resuspended cell nucleus suspension; "Nucleus extraction" refers to the microliters of Nuclei Resuspension Buffe resuspended cell nucleus suspension used for single-nucleus transcription library construction; "Total nucleus count" refers to the total number of cell nuclei obtained by multiplying "Nucleus concentration" by "Nucleus solution"; and "Remaining nuclei" refers to the number of cell nuclei remaining after removing 75,000 cell nuclei used in the single-nucleus transcription library construction, which is the difference between "Total nucleus count" and 75,000.

[0132] 2. RNA extraction and library construction results

[0133] Samples 1 and 2 were cell nuclei remaining after single-cell extraction. After centrifugation and washing with RNase off-H2O, RNA was extracted directly using the QIAamp DNAMini Kit. Samples 3 to 5 were cell nuclei remaining after single-cell extraction. These nuclei were preserved in Trizol LS and RNA was extracted using the Trizol method. The detection results of the extracted RNA are shown in Table 2, and the detection results of the whole transcriptome library are shown in Table 3. Figures 1 to 5 As shown, Figures 1 to 5 The images show the test results for samples 1 through 5 in sequence.

[0134] Table 2. RNA detection results

[0135] sample Concentration (ng / μL) Volume (μL) Total amount (μg) RIN / RQN 28S / 18S Sample1 32.2 9 0.29 3.19 0.51 Sample2 10.6 9 0.095 3.23 0.34 Sample3 48.800 15 0.732 6.250 0.700 Sample4 20.400 15 0.306 4.220 0.670 Sample5 43.000 15 0.645 5.260 0.730

[0136] In Table 2, "RIN / RQN" refers to the RNA integrity number, and "28S / 18S" refers to the ratio of the two major subunits of eukaryotic RNA (ribosomal RNA), 28S and 18S. All values ​​were detected using RNA Qsep400.

[0137] Table 3. Results of whole transcriptome library analysis

[0138] sample Barcode Library concentration (ng / μL) Library volume (μL) Total number of documents in the library (ng) Sample1 427 1.47 20 29.4 Sample2 426 2.11 20 42.2 Sample3 425 2.55 20 51 Sample4 424 2.67 20 53.4 Sample5 423 2.71 20 54.2

[0139] The test results show that both extraction methods are feasible for nuclear RNA extraction, with no significant difference in extraction efficiency, and both are sufficient for RNA library construction. Since the extracted nuclei are mostly mRNA with virtually no 18S rRNA or 28S rRNA, the RIN value and 28S / 18S ratio are almost meaningless. Due to the heterogeneity of tumor samples among different patients, library yields will not be entirely consistent; the number of templates in the same amount of nucleic acid input will vary, therefore, a library yield of 20 ng to 60 ng is considered normal. In conclusion, the experimental results indicate that the remaining nuclei in single-cell transcriptomics are sufficient for nuclear RNA library construction and sequencing.

[0140] 3. Single-cell nucleus RNA sequencing results

[0141] In this experiment, single-cell nuclear transcription libraries were constructed from five samples, and the libraries were sequenced. Sequencing data were analyzed using RiboDetector to remove rRNA reads; Cutadapt was used to remove adapter sequences from reads, remove low-quality bases at the ends of reads, and remove reads containing excessive N; isofox was used to detect gene expression levels and gene fusions. Some results are shown below. Figure 6 and Figure 7 As shown. Figure 6 and Figure 7 The sequencing data analysis results are for sample 1.

[0142] Single-cell transcriptome sequencing results showed that the data quality control was ideal, with a total cell number of 7585, a number of beads per droplet of almost 1, and median RNA feature count (nFeature RNA), absolute UMI count (nCount RNA), and mitochondrial percentage of 140, 240, and 0.5%, respectively.

[0143] 4. Nuclear RNA sequencing

[0144] This experiment statistically analyzed the total sequences (number of sequencing reads), rRNA rate (%) (proportion of rRNA reads), clean rate (%) (proportion of clean reads), uniquely mapped rate (proportion of uniquely mapped reads), gene detected (TPM>1) (number of genes with TPM>1 detected), and pass fusion pairs (number of detected gene fusions) of nuclear RNAseq from five samples. The statistical results are as follows: Figures 8 to 13 As shown

[0145] Figures 8 to 13 The results showed that the quality control of nuclear RNA-seq data was ideal, with rRNA accounting for less than 1%, the effective data rate reaching 99%, the proportion of uniquely aligned reads exceeding 89%, the number of genes with TPM>1 detected exceeding 14,000, and the number of gene fusions detected exceeding 290, which is sufficient for subsequent data analysis. In conclusion, the remaining nuclei in single-cell transcriptomics are sufficient for nuclear RNA library construction and sequencing.

[0146] The detection method of this application can complete the construction of a transcription library using a minimum of 95ng of RNA, with a library yield of 42ng. In contrast, sequencing detection only requires a library yield of 6ng. The minimum starting amount for completing the construction of a transcription library is 20ng, and the minimum starting amount for completing the construction of a single-cell nuclear transcription library is 15ng. Therefore, this method can automate both single-cell transcriptomics and nuclear RNA-seq based on a minimum of 35ng of RNA, while the minimum starting amount for conventional whole transcriptomics detection is 200ng.

[0147] In summary, this method, which isolates single-cell samples from a single-needle aspiration specimen and divides them into at least two parts—one for single-cell nucleic acid detection and the other for whole transcriptome and / or whole genome detection—improves the utilization rate of specimens with limited tissue volume. This expands the scope of such samples beyond single-omics detection and reduces the heterogeneity of different omics resulting from multi-needle aspiration specimens. The method is simple, convenient, and easily scalable. It is understood that the detection method described in this application is not necessarily limited to library construction for nuclear RNA; it can also utilize the cell nucleus for DNA extraction and genomic detection.

[0148] The above description, in conjunction with specific embodiments, provides a further detailed explanation of this application and should not be construed as limiting the specific implementation of this application to these descriptions. Those skilled in the art to which this application pertains can make several simple deductions or substitutions without departing from the concept of this application.

Claims

1. A method for detecting a specimen based on a needle puncture, the method comprising: This includes performing single-cell nucleus extraction on all single-needle puncture specimens to obtain single-cell nucleus samples; ​ A portion of the single-cell nucleus sample is used for single-cell nucleic acid detection, and the remaining single-cell nucleus sample is used directly for whole transcriptome and / or whole genome detection. Alternatively, the remaining single-cell nucleus sample is cryopreserved for use in whole transcriptome and / or whole genome detection.

2. The detection method according to claim 1, characterized in that: The single-cell nucleic acid detection is single-cell nuclear transcriptome sequencing detection and / or single-cell whole genome sequencing detection; Preferably, the single-needle puncture specimen is obtained by sampling with a sampling needle of 16g / 20mm or larger.

3. The detection method according to claim 1, characterized in that: The single-cell nucleus extraction process includes obtaining a single-cell nucleus sample by grinding and separating the puncture tissue obtained from a single needle puncture. Preferably, obtaining single-cell nucleus samples by grinding and separation includes cutting the puncture tissue into small pieces in a homogenization buffer. The tissue block was ground using a tissue homogenizer and filtered through a cell sieve to remove residue and collect the filtrate. The filtrate was centrifuged for the first time and the supernatant was discarded. The precipitate was resuspended in nuclear washing buffer and centrifuged for the second time and the supernatant was discarded. This step was repeated at least once. The precipitate was resuspended in nuclear buffer to obtain the single-cell nucleus sample.

4. The detection method according to claim 3, characterized in that: The cell sieve filtration process includes first filtering with a 70μm to 90μm cell sieve, collecting the filtrate and grinding it again, then filtering with a 30μm to 40μm cell sieve, collecting the filtrate and centrifuging it for the first time.

5. The detection method according to claim 3, characterized in that: The conditions for the first centrifugation were: 4°C, 500g for 5-8 minutes. Preferably, the conditions for the second centrifugation are: 4°C, 800g, centrifugation for 8-10 minutes.

6. The detection method according to claim 3, characterized in that: The homogenization buffer contains 1× basal buffer, 240 mmol / L to 260 mmol / L sucrose, 0.9% to 1.1% BSA, 0.9 mmol / L to 1.1 mmol / L DTT, 1× protease inhibitor mixture, and 0.3 U / μL to 0.5 U / μL ribonuclease inhibitor; The 1× basal buffer contains 18 mmol / L to 22 mmol / L Tris, 22.5 mmol / L to 27.5 mmol / L KCl, and 4.5 mmol / L to 5.5 mmol / L MgCl2.

7. The detection method according to claim 3, characterized in that: The cell nucleus washing solution is a homogenization buffer containing iodixanol; Preferably, the concentration of iodixanol in the cell nucleus washing solution is 20% to 25%.

8. The detection method according to claim 3, characterized in that: The nuclear buffer is a PBS buffer containing 0.9% to 1.1% BSA, and also contains 0.4 U / μL to 0.5 U / μL of ribonuclease inhibitor.

9. A kit for use in the detection method according to any one of claims 1-8, characterized in that: The solution includes 1 mol / L sucrose solution, 6× basal buffer, BSA, 100 mmol / L DTT, a mixture of protease inhibitors, 40 U / μL ribonuclease inhibitor, 60% iodixanol, PBS buffer, and nuclease-free water. The 1 mol / L sucrose solution was obtained by dissolving sucrose in water. The solvent for the 6× basic buffer is nuclease-free water, which contains 120 mmol / L Tris, 150 mmol / L KCl, and 30 mmol / L MgCl2. One tablet of the protease inhibitor mixture is dissolved in 0.5 mL of nuclease-free water to obtain a 100× protease inhibitor mixture.

10. The reagent kit according to claim 9, characterized in that: When using this solution, prepare a homogenization buffer using 6× basic buffer, 1 mol / L sucrose solution, 10% BSA aqueous solution, 100 mmol / L DTT, 100× protease inhibitor mixture, 40 U / μL ribonuclease inhibitor and nuclease-free water. Preferably, a 50% iodixanol solution is prepared using 60% iodixanol and 6× basal buffer, and then a cell nucleus washing solution is prepared using the 50% iodixanol solution and homogenization buffer. Preferably, the nuclear buffer is prepared using PBS buffer containing 10% BSA, 40 U / μL ribonuclease inhibitor, and PBS buffer.

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