Dry blood spot pretreatment method and RNA extraction method

Abstract

The invention provides a dry blood spot pretreatment method and an RNA extraction method.The dry blood spot pretreatment method comprises the steps that lysate is added into to-be-treated dry blood spots for incubation, ultrasonic treatment is carried out so that RNA in the dry blood spots can be released, and a mixed solution containing RNA is obtained; extracting the mixed solution by adopting an extracting agent to obtain a supernatant containing RNA (Ribonucleic Acid); wherein the extraction agent is chloroform or 1-bromo-3-chloropropane.

Description

Technical Field

[0001] The present application relates to a dried blood spot pretreatment method and an RNA extraction method. Background Art

[0002] RNA extraction technology is a key step in molecular biology experiments, which involves isolating RNA from cell or tissue samples for subsequent analysis. It has a wide range of applications in biological and medical research, providing important tools and methods for understanding the regulation of gene expression, disease mechanisms, and drug responses in organisms. Existing RNA extraction technology is generally applicable to samples such as blood, bone marrow fluid, cell culture fluid, and tissues. Summary of the invention

[0003] The present application provides a dried blood spot pretreatment method and an RNA extraction method, using dried blood spots as samples for easy transportation, detection and storage.

[0004] One aspect of the present application provides a dried blood spot pretreatment method, comprising:

[0005] adding a lysis solution to the dried blood spot to be processed for incubation, and performing ultrasonic treatment to release RNA in the dried blood spot to obtain a mixed solution containing RNA;

[0006] extracting the mixed solution with an extractant to obtain a supernatant containing RNA;

[0007] Wherein, the extractant is chloroform or 1-bromo-3-chloropropane.

[0008] Another aspect of the present application provides a dried blood spot pretreatment method, comprising:

[0009] adding a lysis solution and a detergent to the dried blood spot to be processed for incubation, and performing ultrasonic treatment to release RNA in the dried blood spot to obtain a mixed solution containing RNA;

[0010] extracting the mixed solution with an extractant to obtain a supernatant containing RNA;

[0011] Wherein, the extractant is chloroform or 1-bromo-3-chloropropane;

[0012] The detergent contains one or more of sodium lauroyl sarcosinate, sodium lauryl sulfate or Tween-20.

[0013] Another aspect of the present application provides a method for extracting RNA from dried blood spots, comprising:

[0014] adding a lysis solution to the dried blood spot to be processed for incubation, and performing ultrasonic treatment to release RNA in the dried blood spot to obtain a mixed solution containing RNA;

[0015] Extracting the mixed solution with an extractant to obtain a supernatant containing RNA; wherein the extractant is chloroform or 1-bromo-3-chloropropane;

[0016] RNA was extracted from the RNA-containing supernatant.

[0017] Another aspect of the present application provides adding a lysing solution and a detergent to a dried blood spot to be processed for incubation, performing ultrasonic treatment to release RNA in the dried blood spot, and obtaining a mixed solution containing RNA;

[0018] extracting the mixed solution with an extractant to obtain a supernatant containing RNA;

[0019] Wherein, the extractant is chloroform or 1-bromo-3-chloropropane; the detergent contains one or more of sodium lauroyl sarcosinate, sodium dodecyl sulfate or Tween-20;

[0020] RNA was extracted from the RNA-containing supernatant.

[0021] The present application provides a dried blood spot pretreatment method and an RNA extraction method. The dried blood spot pretreatment method of the present application is used to separate the supernatant containing RNA, and then RNA can be extracted from the dried blood spot for subsequent testing, such as quantitative detection of gene expression. The present application uses dried blood spots as samples, which is convenient for transportation, detection and storage, and also broadens the scope of application of dried blood spots. Since dried blood spots are dried whole blood, the amount of sample required for RNA extraction from dried blood spots is also small. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 The integrity results of sample 1 are shown;

[0023] Figure 2 The integrity results of sample 2 are shown;

[0024] Figure 3 The integrity results of sample 3 are shown;

[0025] Figure 4 The integrity results of sample 4 are shown;

[0026] Figure 5 The integrity results of sample 5 are shown;

[0027] Figure 6 The integrity results of sample 6 are shown;

[0028] Figure 7 The integrity results of sample 7 are shown;

[0029] Figure 8 The integrity results for Sample 8 are shown. DETAILED DESCRIPTION

[0030] Here, the technical solutions in the embodiments (or "implementations") of the present application will be described clearly and completely in conjunction with the accompanying drawings. When the following description refers to the drawings, unless otherwise indicated, the same numbers in different drawings represent the same or similar elements. The terms "first" and "second" in the embodiments of the present application are only used for the purpose of convenience of description and cannot be understood as indicating or implying relative importance.

[0031] The present application provides a method for pretreatment of dried blood spots. Dried blood spots are a method for collecting whole blood samples on filter paper and drying them to form dried blood plaques through dried blood sampling technology. Without any additional treatment, the analytes and blood components can be adsorbed on a solid cellulose matrix, which has strong stability. At present, dried blood spots are mainly subjected to solvent extraction, and tandem mass spectrometry and other methods are used to detect and analyze the components to be detected in the dried blood spots.

[0032] One aspect of the present application is a method for pre-treating a dried blood spot, comprising;

[0033] adding a lysis solution to the dried blood spot to be processed for incubation, performing ultrasonic treatment (promoting RNA release through cavitation effect and mechanical vibration) to release RNA in the dried blood spot, and obtaining a mixed solution containing RNA;

[0034] extracting the mixed solution with an extractant to obtain a supernatant containing RNA;

[0035] Wherein, the extractant is chloroform or 1-bromo-3-chloropropane; or

[0036] adding a lysis solution and a detergent to the dried blood spot to be processed for incubation, and performing ultrasonic treatment to release RNA in the dried blood spot to obtain a mixed solution containing RNA;

[0037] extracting the mixed solution with an extractant to obtain a supernatant containing RNA;

[0038] Wherein, the extractant is chloroform or 1-bromo-3-chloropropane;

[0039] The detergent contains one or more of sodium lauroyl sarcosinate, sodium lauryl sulfate or Tween-20.

[0040] The present application uses dried blood spots as samples, which is convenient for transportation, detection and storage, and also broadens the application scope of dried blood spots. Since dried blood spots are dried whole blood, the amount of sample required for extracting RNA from dried blood spots is also small.

[0041] Compared with chloroform, 1-bromo-3-chloropropane has high volatility and toxicity. Inhalation of high concentrations of chloroform gas can cause poisoning symptoms such as dizziness, fatigue, nausea, and vomiting. Therefore, when using chloroform as an extraction agent, it is necessary to strictly abide by the chemical experiment operation procedures and strengthen safety measures such as ventilation protection to avoid safety accidents. The extraction agent is 1-bromo-3-chloropropane, which has relatively low toxicity and is more convenient to operate.

[0042] Specific details can be found in the following embodiments:

[0043] All products marked with an asterisk * in the following text are commercial products in the QIAGEN (brand name) kit

[0044] OD value: Calculated by measuring the absorbance of RNA solution at 260 nm and 280 nm. Usually a ratio between 1.8-2.0 is more appropriate.

[0045] RQN value: can be used to evaluate the quality of RNA. The software calculates the RNA quality number (RQN) and gives an RQN value between 1 and 10 based on the integrity of 18S and 28S and the peak area ratio. The higher the RQN value, the better the RNA integrity, that is, the higher the RNA quality.

[0046] QIAzol Lysis Buffer: QIAGEN, Catalog No.: 5346994, Lot No. 57502096

[0047] Sodium dodecyl sulfate: sigma Product number: L6026 Batch number: SLBZ2585

[0048] Sodium lauroyl sarcosinate: sigma Product number: 61743 Batch number: BCCG4842

[0049] Tween-20: Sigma Part Number: V900548, Batch Number: WXBC9582V

[0050] Chloroform: GENERAL-REAGENT Product Number: G75915B, Lot Number: P1456081

[0051] 1-Bromo-3-chloropropane: MACKLIN Product number: B802344, Batch number: C16492206

[0052] Anhydrous ethanol: Sigma Part No.: 64175, Batch No.: 2022101202

[0053] EDTA: sigma Product number: E6758, Lot number: SLBR6878V

[0054] Tris-HCl: sigma Product number: T3253, Lot number: SLBX8307

[0055] RNeasy Plus Universal Mini Kit: QIAGEN Catalog No.: 1062832, Lot No. 175031075

[0056] Example 1 Pretreatment of dried blood spots

[0057] Use a hole puncher to cut two blood spots from the dried blood spot storage card, place them in a 2 ml test tube, and then add 1 ml QIAzol lysis buffer (QIAGEN, batch number 57502096); incubate the test tube at 37°C with shaking at 450 rpm for 15 minutes, centrifuge for 8 seconds, and then perform ultrasonic treatment at room temperature for 15 minutes; incubate at 37°C with shaking at 450 rpm for 15 minutes again; then add 0.25 ml chloroform and shake vigorously twice, let stand at room temperature for 5 minutes, and then centrifuge at 12000 rpm for 15 minutes at 4°C; then take 0.45 ml of supernatant, transfer to a 2 ml test tube, add 0.8 ml ethanol, and mix thoroughly with a pipette to obtain sample 1.

[0058] Example 2 Pretreatment of dried blood spots

[0059] Two blood spots were cut from the dried blood spot storage card using a hole puncher and placed in a 2 ml test tube. Then, 1 ml of QIAzol lysis buffer (QIAGEN, batch number 57502096) and 100 μl of 0.2 g / ml sodium lauroyl sarcosinate detergent (1 g sodium lauroyl sarcosinate, 0.03653 g ethylenediaminetetraacetic acid (EDTA) dissolved in 5 ml Tris-HCl solution) were added. The test tube was incubated at 37°C with shaking at 450 rpm for 15 minutes, centrifuged for 8 seconds, and then sonicated at room temperature for 15 minutes. The tube was incubated at 37°C with shaking at 450 rpm for 15 minutes. Then, 0.25 ml of chloroform was added and vigorously shaken twice. After standing at room temperature for 5 minutes, the tube was centrifuged at 12,000 rpm for 15 minutes at 4°C. Then, 0.45 ml of the supernatant was taken and transferred to a 2 ml test tube. 0.8 ml of ethanol was added and the tube was fully mixed using a pipette to obtain sample 2.

[0060] Example 3 Pretreatment of dried blood spots

[0061] Two blood spots were cut from the dried blood spot storage card using a hole puncher and placed in a 2 ml test tube. Then, 1 ml of QIAzol lysis buffer (QIAGEN, batch number 57502096) and 100 μl of 0.2 g / ml sodium dodecyl sulfate (SDS) detergent (1 g of sodium dodecyl sulfate, 0.03653 g of ethylenediaminetetraacetic acid (EDTA) dissolved in 5 ml of Tris-HCl solution) were added. The test tube was incubated at 37°C with shaking at 450 rpm for 15 minutes, centrifuged for 8 seconds, and then sonicated at room temperature for 15 minutes. The tube was incubated at 37°C with shaking at 450 rpm for 15 minutes. Then, 0.25 ml of chloroform was added and the tube was shaken vigorously twice. After standing at room temperature for 5 minutes, the tube was centrifuged at 12,000 rpm for 15 minutes at 4°C. Then, 0.45 ml of the supernatant was taken and transferred to a 2 ml test tube. 0.8 ml of ethanol was added and the tube was mixed thoroughly using a pipette to obtain sample 3.

[0062] Example 4 Pretreatment of dried blood spots

[0063] Two blood spots were cut from the dried blood spot storage card using a hole puncher and placed in a 2 ml test tube. Then, 1 ml of QIAzol lysis buffer (QIAGEN, batch number 57502096) and 100 μl of 0.2 ml / ml Tween-20 detergent (1 ml of Tween-20, 0.03653 g of ethylenediaminetetraacetic acid (EDTA) dissolved in 4 ml of Tris-HCl solution) were added. The test tube was incubated at 37°C with shaking at 450 rpm for 15 minutes, centrifuged for 8 seconds, and then sonicated at room temperature for 15 minutes. The tube was incubated at 37°C with shaking at 450 rpm for 15 minutes. Then, 0.25 ml of chloroform was added and vigorously shaken twice. After standing at room temperature for 5 minutes, the tube was centrifuged at 12,000 rpm for 15 minutes at 4°C. Then, 0.45 ml of the supernatant was taken and transferred to a 2 ml test tube. 0.8 ml of ethanol was added and the tube was mixed thoroughly using a pipette to obtain sample 4.

[0064] Example 5 Pretreatment of dried blood spots

[0065] Use a hole puncher to cut two blood spots from the dried blood spot storage card, place them in a 2 ml test tube, and then add 1 ml QIAzol lysis buffer (QIAGEN, batch number 57502096); incubate the test tube at 37°C with shaking at 450 rpm for 15 minutes, centrifuge for 8 seconds, and then perform ultrasonic treatment at room temperature for 15 minutes; incubate at 37°C with shaking at 450 rpm for 15 minutes; then add 0.25 ml 1-bromo-3-chloropropane and shake vigorously twice, let stand at room temperature for 5 minutes, and then centrifuge at 12000 rpm for 15 minutes at 4°C; then take 0.45 ml of supernatant, transfer it to a 2 ml test tube, add 0.8 ml ethanol, and mix thoroughly with a pipette to obtain sample 5.

[0066] Example 6 Pretreatment of Dried Blood Spots

[0067] Use a hole puncher to cut two blood spots from the dried blood spot storage card, place them in a 2 ml test tube, then add 1 ml QIAzol lysis buffer (QIAGEN, batch number 57502096) and 100 μl 0.2 g / ml sodium lauroyl sarcosinate detergent; incubate the test tube at 37°C with shaking at 450 rpm for 15 minutes, centrifuge for 8 seconds, and then perform ultrasonic treatment at room temperature for 15 minutes; incubate again at 37°C with shaking at 450 rpm for 15 minutes; then add 0.25 ml 1-bromo-3-chloropropane and shake vigorously twice, let stand at room temperature for 5 minutes, and then centrifuge at 12000 rpm for 15 minutes at 4°C; then take 0.45 ml of supernatant, transfer it to a 2 ml test tube, add 0.8 ml ethanol, and mix thoroughly with a pipette to obtain sample 6.

[0068] Example 7 Pretreatment of Dried Blood Spots

[0069] Use a hole puncher to cut two blood spots from the dried blood spot storage card, place them in a 2 ml test tube, then add 1 ml QIAzol lysis buffer (QIAGEN, batch number 57502096) and 100 μl 0.2 g / ml sodium dodecyl sulfate (SDS) detergent; incubate the test tube at 37°C with shaking at 450 rpm for 15 minutes, centrifuge for 8 seconds, and then perform ultrasonic treatment at room temperature for 15 minutes; incubate again at 37°C with shaking at 450 rpm for 15 minutes; then add 0.25 ml 1-bromo-3-chloropropane and shake vigorously twice, let stand at room temperature for 5 minutes, and then centrifuge at 12000 rpm for 15 minutes at 4°C; then take 0.45 ml of supernatant, transfer to a 2 ml test tube, add 0.8 ml ethanol, and mix thoroughly with a pipette to obtain sample 7.

[0070] Example 8 Pretreatment of Dried Blood Spots

[0071] Use a hole puncher to cut two blood spots from the dried blood spot storage card, place them in a 2 ml test tube, then add 1 ml QIAzol lysis buffer (QIAGEN, batch number 57502096) and 100 μl 0.2 ml / ml Tween-20 detergent; incubate the test tube at 37°C with shaking at 450 rpm for 15 minutes, centrifuge for 8 seconds, and then perform ultrasonic treatment at room temperature for 15 minutes; incubate at 37°C with shaking at 450 rpm for 15 minutes again; then add 0.25 ml 1-bromo-3-chloropropane and shake vigorously twice, let stand at room temperature for 5 minutes, and then centrifuge at 12000 rpm for 15 minutes at 4°C; then take 0.45 ml of supernatant, transfer it to a 2 ml test tube, add 0.8 ml ethanol, and mix thoroughly using a pipette to obtain sample 8.

[0072] Example 9 RNA extraction

[0073] Add RNA kit to samples 1-8 respectively for treatment, take 0.7 ml of the mixed solution, transfer to RNeasyMini column*, centrifuge at 8000 rpm for 30 seconds, and discard the lower solution; take the remaining mixed solution and add to the same RNeasyMini column*, centrifuge at 8000 rpm for 30 seconds again, and discard the lower solution;

[0074] Add 700 μl of buffer RWT* to the RNeasy Mini column*, centrifuge at 8000 rpm for 30 seconds, and discard the lower solution; add 500 μl of buffer RPE* to the RNeasy Mini column*, centrifuge at 8000 rpm for 30 seconds, and discard the lower solution (repeat this step twice);

[0075] Then centrifuge at 8000rpm for 2 minutes, transfer the RNeasy Mini* to a 1.5ml test tube, and add 50μl RNase-free water*; then incubate at room temperature for 1 minute, and then centrifuge at 8000rpm for 1 minute; remove the RNeasy mini column and transfer the freshly extracted RNA solution to a 1.5ml test tube;

[0076] Finally, the RNA concentration was measured by a spectrophotometer (NanoDrop One). The OD value (A260 / A280) was calculated by the absorbance of the RNA solution at 260 nm and 280 nm. The RNA quality index (RQN) value of the RNA solution was measured by a fragment analyzer (Bioptic Qsep1).

[0077] Table 1

[0078] Extraction agent Detergent Concentration (ng / uL) OD RQN Example 1 Chloroform none 6.701 1.845 7.42 Example 5 1-Bromo-3-chloropropane none 10.593 1.871 7.41

[0079] Table 1 Figure 1 and Figure 5 The results showed that the OD values ​​of the samples of Example 1 and Example 5 were both within the range of 1.8-2.0, the RQN values ​​were both greater than 7, the RNA was relatively intact, and the concentration of the sample of Example 5 was higher.

[0080] Table 2

[0081]

[0082] Combined with Table 2, Figures 1 to 8 , the OD values ​​of Examples 1 to 8 all fall within the range of 1.8-2.0, and the RQN values ​​are all greater than 7. The concentrations of the samples of Examples 4 and 7 are relatively high, but the RQN values ​​are not significantly different from those of Examples 1 and 8. The concentrations and RQN values ​​of the samples of Examples 2 and 6 are relatively high compared to the other examples. This indicates that the combination of QIAzol lysate, SDS / Tween-20, and chloroform / 1-bromo-3-chloropropane achieves better results in terms of concentration; the combination of QIAzol lysate, sodium lauroyl sarcosinate, and chloroform / 1-bromo-3-chloropropane achieves better results in terms of concentration and integrity. The combination of QIAzol lysate, SDS / Tween-20, and 1-bromo-3-chloropropane and the combination of QIAzol lysate, sodium lauroyl sarcosinate, and 1-bromo-3-chloropropane are more advantageous because 1-bromo-3-chloropropane is less toxic than chloroform.

[0083] It should be noted that the technical solutions or technical features described in the above embodiments can be combined or supplemented with each other without causing conflicts. The scope of protection of this application is not limited to the above embodiments and the drawings; any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of this application shall be included in the scope of protection of this application.

Claims

1. A dried blood spot pretreatment method, comprising: adding a lysis solution to the dried blood spot to be processed for incubation, and performing ultrasonic treatment to release RNA in the dried blood spot to obtain a mixed solution containing RNA; extracting the mixed solution with an extractant to obtain a supernatant containing RNA; Wherein, the extractant is chloroform or 1-bromo-3-chloropropane.

2. The dried blood spot pretreatment method according to claim 1, characterized in that: The lysis buffer is QIAzol.

3. A dried blood spot pretreatment method comprising: adding a lysis solution and a detergent to the dried blood spot to be processed for incubation, and performing ultrasonic treatment to release RNA in the dried blood spot to obtain a mixed solution containing RNA; extracting the mixed solution with an extractant to obtain a supernatant containing RNA; Wherein, the extractant is chloroform or 1-bromo-3-chloropropane; The detergent contains one or more of sodium lauroyl sarcosinate, sodium lauryl sulfate or Tween-20.

4. The dried blood spot pretreatment method according to claim 3, characterized in that: The lysis buffer is QIAzol.

5. The dried blood spot pretreatment method according to claim 3, characterized in that: The detergent is an EDTA-Tris-HCl solution containing 0.2 g / ml sodium lauroyl sarcosinate, 0.2 g / ml sodium dodecyl sulfate or 0.2 ml / ml Tween-20.

6. A method for extracting RNA from dried blood spots, comprising: adding a lysis solution to the dried blood spot to be processed for incubation, and performing ultrasonic treatment to release RNA in the dried blood spot to obtain a mixed solution containing RNA; Extracting the mixed solution with an extractant to obtain a supernatant containing RNA; wherein the extractant is chloroform or 1-bromo-3-chloropropane; RNA was extracted from the RNA-containing supernatant.

7. The method for extracting RNA according to claim 6, wherein The lysis buffer is QIAzol.

8. A method for extracting RNA from dried blood spots, comprising: adding a lysis solution and a detergent to the dried blood spot to be processed for incubation, and performing ultrasonic treatment to release RNA in the dried blood spot to obtain a mixed solution containing RNA; extracting the mixed solution with an extractant to obtain a supernatant containing RNA; Wherein, the extractant is chloroform or 1-bromo-3-chloropropane; the detergent contains one or more of sodium lauroyl sarcosinate, sodium dodecyl sulfate or Tween-20; RNA was extracted from the RNA-containing supernatant.

9. The method for extracting RNA according to claim 7, characterized in that, The lysis buffer is QIAzol.

10. The method for extracting RNA according to claim 7, characterized in that: The detergent is an EDTA-Tris-HCl solution containing 0.2 g / ml sodium lauroyl sarcosinate, 0.2 g / ml sodium dodecyl sulfate or 0.2 ml / ml Tween-20.

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