A method for correcting centrifugal force deviation during sample separation

Abstract

The present invention discloses a method for correcting centrifugal force deviation during sample separation, comprising the following steps: 1) adding a gel-like substance to a centrifuge tube of a target size, placing a cone above the gel-like substance with its tip facing the gel, and serving as a standard centrifuge tube; preparing several centrifuge tubes in parallel using the same method, raw materials, and standards, and serving as correction centrifuge tubes; 2) centrifuging the standard centrifuge tubes in a standard centrifuge under standard conditions, and marking the depth at which the cones are immersed in the gel; 3) centrifuging the correction centrifuge tubes in a centrifuge to be corrected under standard conditions, adjusting the centrifugation conditions according to the depth at which the cones are immersed in the gel until the depth at which the cones are immersed in the gel is the same as the depth at which the cones are immersed in the gel in step 2), and recording the correction centrifugation conditions; and 4) centrifuging the sample in the centrifuge to be corrected according to the correction centrifugation conditions, thereby correcting the centrifugal force deviation during sample separation. This method can correct centrifugal force deviation and ensure the accuracy of sample separation.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and in particular relates to a method for correcting centrifugal force deviation in a sample separation process. Background Art

[0002] Centrifugal force is one of the key factors affecting experiments. Due to the differences in actual centrifugal force between different laboratories, brands, and models, the centrifugal force varies from one centrifuge to another, resulting in different experimental results for the same test kit when used in different centrifuges.

[0003] Differences in the speed ramp-up and down times of centrifuges from different manufacturers can lead to deviations in the cumulative centrifugation time during sample separation. The centrifugal force indicated on the centrifuge is usually calculated based on the maximum outer diameter of the compatible rotor, so deviations in the sizes of centrifuge tubes compatible with the centrifuge rotor can cause deviations in the centrifugal force during sample separation. Basket-type centrifuges and rotor-type centrifuges can also experience centrifugal force deviations during sample separation. Deviations in centrifugal force can result in differences in the target centrifugal components of the sample after centrifugation, leading to deviations in sample measurements during subsequent sample testing. In particular, this effect can accumulate in multi-step centrifugation experiments. Summary of the Invention

[0004] Purpose of the invention: To solve the above technical problems, the present invention provides a method for correcting centrifugal force deviation during sample separation.

[0005] Technical solution: In order to achieve the above-mentioned purpose, the present invention adopts the following technical solution:

[0006] A method for correcting centrifugal force deviation during sample separation comprises the following steps:

[0007] 1) Add a gel-like substance to a centrifuge tube of the target size, and place a cone on top of the gel-like substance with its tip facing the gel to serve as a standard centrifuge tube. Prepare several centrifuge tubes in parallel using the same method, materials, and standards to serve as calibration centrifuge tubes.

[0008] 2) Centrifuge the standard centrifuge tube in a standard centrifuge under standard conditions, marking the depth at which the cone is immersed in the gel;

[0009] 3) Centrifuge the calibration centrifuge tube in the centrifuge to be calibrated under standard conditions, adjusting the centrifugation conditions according to the depth of the cone immersed in the gel until the depth of the cone immersed in the gel is the same as the depth of the cone immersed in the gel in step 2), and record the calibration centrifugation conditions;

[0010] 4) Centrifuge the sample in the centrifuge to be calibrated according to the calibration centrifugation conditions, thereby correcting the centrifugal force deviation during the sample separation process.

[0011] As a specific embodiment, in step 1), the target size centrifuge tube is determined according to the centrifuge model to be calibrated, and is selected from 0.2, 0.3, 0.5, 1.5, 2, 5, 7, 10, 12, 15, 20, 26.3, 30, 32.4, 35, 50, 85 or 100 mL.

[0012] As a specific embodiment, in step 1), the gel-like substance is selected from agarose gel, agar, acrylamide gel, silica gel, gelatin, aerogel, carrageenan, xanthan gum or glucomannan; preferably agarose gel.

[0013] As a specific embodiment, in step 1), the volume of the gel-like substance added depends on the size of the centrifuge tube. The liquid level should be within the range of 1 / 20-19 / 20 of the tube depth, preferably 1 / 2 of the tube depth. When the gel liquid level is vertical in the centrifuge tube, the liquid level should be horizontal. The gel volume in centrifuge tubes from the same batch should be consistent.

[0014] As a specific embodiment, in step 1), the concentration of the gel-like substance ranges from 0.1% to 50%. The specific concentration used depends on the type of gel and the desired centrifugation time and speed. The longer the centrifugation time or the higher the speed, the higher the gel concentration. The gel concentration should be consistent across centrifuge tubes from the same batch.

[0015] As a specific implementation scheme, in step 1), the cone refers to a shape with a round and thick upper part and a sharp lower part; the cone is hollow or solid; the cross-section of the upper part of the cone is the same as or slightly smaller than the cross-sectional area of ​​the centrifuge tube, preferably a cylindrical shape with the same cross-sectional area as the centrifuge tube, and has an air guide groove to ensure that the cone remains upright when placed in the centrifuge tube without suffocation; the maximum range of the cross-sectional area of ​​the cone can be equivalent to the inner diameter of the centrifuge tube used, and the minimum diameter is 1 mm, such as Figure 1 Specific cone parameters should be determined based on experimental conditions. The cone height should be less than the distance from the gel surface to the centrifuge tube opening. The cone material should be a combination of one or more materials: plastic, metal, or glass. Cone weight should be consistent within a batch. Cone weight depends on the type of gel, the centrifugation time, and the speed required for evaluation. Higher gel concentrations, longer centrifugation times, or higher speeds result in lighter cone weight.

[0016] As a specific embodiment, the gel-like substance and cone-shaped object in step 1) are selected by centrifuging in a standard centrifuge under standard conditions according to the method in step 2), and the tip of the cone is completely immersed in the gel, preferably immersed in the gel to a depth of 1 / 3-2 / 3, and the cone is not completely immersed in the gel.

[0017] As a specific implementation scheme, in step 2), the standard conditions in the standard centrifuge refer to the centrifuge and centrifugal conditions used in the target experiment, and the centrifugal force under the centrifugal conditions needs to be reproduced on other centrifuges.

[0018] As a specific implementation scheme, in step 3), the centrifugation conditions include centrifugal force, centrifugation time, acceleration rate and deceleration rate.

[0019] Beneficial effects: The present invention can quickly and conveniently evaluate the centrifugal force during the sample separation process according to the depth of the cone immersed in the gel, and can be used to fine-tune the rotation speed and time to ultimately keep the centrifugal force consistent with the standard value, thereby correcting the centrifugal force deviation during the sample separation process and ensuring the accuracy of sample separation. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 : cone cross-section range;

[0021] Figure 2 : Setting of gel-like substance and cone (taking 50mL centrifuge tube as an example);

[0022] Figure 3 : Schematic diagram of the flow chart of the method for correcting centrifugal force deviation in Example 1. DETAILED DESCRIPTION

[0023] The following is a comprehensive description of the present invention. The implementation cases described are the most preferred implementation methods of the present invention, but the present invention is not limited to the following examples.

[0024] Example

[0025] 1. Prepare two centrifuges of different manufacturers and models suitable for 15 mL centrifuge tubes. The centrifugal speed increases and decreases quickly, the centrifugal force is standard, and it is a centrifuge commonly used in this laboratory. Name it Centrifuge A; the centrifugal speed increases and decreases slowly, and the overall speed increase and decrease time is 5 minutes longer than that of Centrifuge A. Name it Centrifuge B.

[0026] 2. Add 8 mL of 1% agarose gel to a 15 mL centrifuge tube;

[0027] 3. Place a cone with a maximum diameter of 10 mm, a minimum diameter of 1 mm, a length of 25 mm, and a mass of 0.2 g on top of the gel, with its tip facing the gel and an air guide groove between the upper round thick part and the inner wall of the centrifuge tube. Figure 2 Design: Cone made of plastic.

[0028] 4. In centrifuge A, centrifuge for 10 minutes at 1,300 g centrifugal force, with an acceleration level of 7 and a deceleration level of 1.

[0029] 0 conditions and centrifuged. After centrifugation, the marked cone was immersed in the gel to a depth of 10 mm;

[0030] 5. In centrifuge B, centrifuge the gel and cone tubes prepared in parallel at 1,300 g for 10 min, with an acceleration level of 7 and a deceleration level of 0, until the cone is immersed in the gel to a depth of 15 mm.

[0031] 6. Based on the difference in the depth of the cone immersed in the gel after centrifugation, fine-tune the centrifugation time of centrifuge B by 30 seconds and the depth of the cone immersed in the gel by 10 mm to make the centrifugal force of the two centrifuges consistent. The corrected centrifugation conditions are recorded as follows: 1,300 g centrifugal force, 9.5 minutes of centrifugation, acceleration level 7, and deceleration level 0.

[0032] 7. Take three freshly collected whole blood samples. Centrifuge the first one using Centrifuge A at 1,300 g for 10 min, with an acceleration level of 7 and a deceleration level of 0 to separate the plasma. This is designated Sample 1.

[0033] 8. The second centrifuge was used with the fine-tuned centrifuge B, with a recorded centrifugal force of 1,300 g, for 9.5 min, an ascending speed level 7, and a descending speed level 0, to separate the plasma, designated as Sample 2.

[0034] 9. Use centrifuge B for the third centrifuge, using the indicated centrifugal force of 1,300 g for 10 min, with an acceleration level of 7 and a deceleration level of 0, to separate the plasma, designated as Sample 3.

[0035] 10. Plasma proteome was extracted and tested from the three separated plasmas. The results are shown below:

[0036] Number of protein identifications Number of peptide identifications Sample 1 3755 35085 Sample 2 3689 34879 Sample 3 3163 29734

[0037] The number of protein and peptide identifications in sample 3 was 15% lower than that in samples 1 and 2. After centrifugation with centrifugal force calibration, the number of protein and peptide identifications in sample 2 was comparable to that in sample 1.

Claims

1. A method for correcting centrifugal force deviation during sample separation, characterized in that: The following steps are involved: 1) Add gel-like substance to a centrifuge tube of target size. Place a cone on top of the gel-like substance with its tip facing the gel to serve as a standard centrifuge tube. Prepare several centrifuge tubes in parallel using the same method, materials, and standards to serve as calibration centrifuge tubes. 2) Centrifuge the standard centrifuge tubes in a standard centrifuge under standard conditions, marking the depth at which the cones are immersed in the gel. 3) Centrifuge the calibration tube in the centrifuge to be calibrated under standard conditions. Adjust the centrifugation conditions according to the depth of the cone immersed in the gel until the depth of the cone immersed in the gel is the same as the depth of the cone immersed in the gel in step 2). Record the calibration centrifugation conditions. 4) Centrifuge the sample in the centrifuge to be calibrated according to the calibration centrifugation conditions to correct the centrifugal force deviation during the sample separation process.

2. The method for correcting centrifugal force deviation during sample separation according to claim 1, characterized in that: In step 1), the target size of the centrifuge tube is determined according to the centrifuge model to be calibrated, and is selected from 0.2, 0.3, 0.5, 1.5, 2, 5, 7, 10, 12, 15, 20, 26.3, 30, 32.4, 35, 50, 85 or 100 mL.

3. The method for correcting centrifugal force deviation during sample separation according to claim 1, characterized in that: In step 1), the gel-like substance is selected from agarose gel, agar, acrylamide gel, silica gel, gelatin, aerogel, carrageenan, xanthan gum or glucomannan.

4. The method for correcting centrifugal force deviation during sample separation according to claim 1, characterized in that: In step 1), the volume of the gel-like substance added depends on the size of the centrifuge tube, and the liquid level should be within the range of 1 / 20-19 / 20 of the tube depth; when the gel liquid level in the centrifuge tube is vertical, the liquid level needs to be horizontal.

5. The method for correcting centrifugal force deviation during sample separation according to claim 1, characterized in that: In step 1), the concentration of the gel-like substance ranges from 0.1% to 50%. The specific concentration used is related to the type of gel and the required evaluation centrifugation time and speed. The longer the centrifugation time or the higher the speed, the higher the gel concentration needs to be.

6. The method for correcting centrifugal force deviation during sample separation according to claim 1, characterized in that: In step 1), the cone is a shape having a rounded upper portion and a pointed lower portion; the cone may be hollow or solid; the cross-section of the cone's upper portion is the same as or slightly smaller than the cross-sectional area of ​​the centrifuge tube, and an air guide groove is provided to ensure that the cone remains upright in the centrifuge tube without suffocating the air; the cone's height is less than the height from the gel liquid surface to the centrifuge tube opening; and the cone may be made of one or more of plastic, metal, or glass.

7. The method for correcting centrifugal force deviation during sample separation according to claim 1, characterized in that: The gel-like substance and the cone-shaped object in step 1) are selected by centrifuging in a standard centrifuge under standard conditions according to the method in step 2) until the tip of the cone is completely immersed in the gel.

8. The method for correcting centrifugal force deviation during sample separation according to claim 1, characterized in that: In step 3), the centrifugation conditions include centrifugal force, centrifugation time, acceleration rate and deceleration rate.

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