CN117821447A

CN117821447A - Extraction kit and extraction method for extracting blood RNA by magnetic bead method

Info

Publication number: CN117821447A
Application number: CN202410117389.1A
Authority: CN
Inventors: 郭元媛; 晋莲; 郭仁康; 潘超; 宋建军
Original assignee: Wuhan Naci Biotechnology Co ltd
Current assignee: Wuhan Naci Biotechnology Co ltd
Priority date: 2024-01-29
Filing date: 2024-01-29
Publication date: 2024-04-05

Abstract

The invention relates to an extraction kit for extracting blood RNA by a magnetic bead method and an extraction method, wherein the kit comprises a lysis binding solution and a magnetic bead washing solution; the lysis binding solution comprises 1-6M guanidine hydrochloride, 10-100mM thiourea, 10-100mM sodium citrate, 1-50mM EDTA, 10-70% isopropyl alcohol, 0.1-20mM sodium chloride, 10-300mM sodium acetate, 0.1-5% hexadecyl sodium sarcosinate and 0.1-5% sodium dodecyl sulfonate; the magnetic bead washing liquid comprises silicon-based magnetic beads and 55% -85% alcohol washing liquid, wherein hydroxyl or carboxyl is modified on the surfaces of the silicon-based magnetic beads, the particle size of the magnetic beads is 20-400nm, and the content of the magnetic beads is 0.5-2mg/mL. The kit can directly extract RNA in cold-stored or frozen blood without the complicated steps of firstly cracking red blood cells, separating, precipitating and collecting white blood cells, and the extracted RNA product has no genome DNA and small fragment DNA/RNA _。

Description

Extraction kit and extraction method for extracting blood RNA by magnetic bead method

Technical Field

The invention relates to the technical field of molecular biology nucleic acid extraction, in particular to an extraction kit and an extraction method for extracting blood RNA by a magnetic bead method.

Background

In modern biological research, RNA is a key to understanding gene expression patterns, regulatory mechanisms, and their responses to environmental changes. With the rapid development of molecular biology and genomics, research on genome level has important effects on the fields of genetic development, epidemic detection, pathological mechanism research, differential diagnosis of microorganisms and the like, and biological research has increasingly demanded high-quality blood RNA. Common traditional methods for extracting blood RNA mainly comprise phenol-chloroform extraction method, guanidine thiocyanate-phenol extraction method and the like, but harmful reagents such as phenol, chloroform or beta-mercaptoethanol are required to be used in the operation process of the methods, so that the methods are harmful to the health of experimental staff.

With the continuous development of biotechnology, many biotechnology companies are conducting research on RNA extraction, and at present, a commonly used kit in RNA extraction mainly comprises a TRIzol reagent one-step method, a TRIzol LS reagent one-step method and a column membrane method, wherein the former two methods use phenol and guanidine isothiocyanate single-phase solutions, so that the health of experimenters is not facilitated, and the extracted blood RNA contains small fragments of impurities such as incompletely degraded DNA/RNA or genomic DNA; the latter needs to lyse red blood cells first, collect white blood cells through centrifugation and then extract, the steps are complicated, the time is 30-120 minutes more, and only fresh blood samples can be extracted; some companies have also proposed kits for direct extraction of non-fresh blood samples, however such kits are of a smaller variety and of uneven quality on the market. The patent CN 114350650A contains erythrocyte lysate, and when blood RNA is extracted, erythrocyte is removed by centrifugation to precipitate leucocytes, and then the erythrocyte is extracted, but the blood is frozen or stored for too long, so that the hemolysis phenomenon can occur, all cells are broken, the leucocytes can not be separated, and the kit is not suitable for extracting RNA from blood frozen for a long time.

Although the RNA extraction kit and the RNA extraction method of the patent CN 114350654A can directly extract RNA in a blood sample without cracking and centrifuging red blood cells, a small amount of genomic DNA is still contained in the extracted blood RNA. Therefore, research on a blood RNA extraction kit which has no DNA residue, complete structure and high purity is of great significance, especially the kit can directly extract RNA in refrigerated or frozen blood samples.

Disclosure of Invention

Aiming at the above defects or improvement demands of the prior art, the invention provides an extraction kit and an extraction method for extracting blood RNA by a magnetic bead method, and aims to discover that RNA in a whole blood sample can be directly extracted without firstly cracking red blood cells to collect white blood cell precipitates by combining magnetic beads, and no genomic DNA and small-fragment undegraded complete DNA/RNA residues exist in an extracted RNA product, so that the technical problems that the genomic DNA or the undegraded complete small-fragment DNA/RNA residues are easy to remain in the extraction of blood RNA by the existing commercial RNA extraction kit or the existing commercial RNA extraction kit is mixed with the undegraded complete small-fragment DNA/RNA are solved.

In order to achieve the above object, according to one aspect of the present invention, there is provided an extraction kit for extracting blood RNA by a magnetic bead method, comprising a lysis binding solution and a magnetic bead washing solution;

the lysis binding solution comprises 1-6M guanidine hydrochloride, 10-100mM thiourea, 10-100mM sodium citrate, 1-50mM EDTA, 10-70% isopropyl alcohol, 0.1-20mM sodium chloride, 10-300mM sodium acetate, 0.1-5% hexadecyl sodium sarcosinate and 0.1-5% sodium dodecyl sulfonate;

the magnetic bead washing liquid comprises silicon-based magnetic beads and 55% -85% alcohol washing liquid, wherein hydroxyl or carboxyl is modified on the surfaces of the silicon-based magnetic beads, the particle size of the magnetic beads is 20-400nm, and the content of the magnetic beads is 0.5-2mg/mL.

Preferably, the magnetic bead method is used for extracting blood RNA, the extraction kit is used for extracting chilled or frozen blood, and the lysis binding solution comprises 1-5M guanidine hydrochloride, 10-80mM thiourea, 10-80mM sodium citrate, 1-40mM EDTA, isopropanol with the mass concentration of 20% -70%, 0.1-20mM sodium chloride, 10-300mM sodium acetate, hexadecyl sodium sarcosinate with the mass concentration of 0.1% -4% and sodium dodecyl sulfonate with the mass concentration of 0.1% -4%.

Preferably, the magnetic bead method is used for extracting blood RNA, the magnetic bead washing liquid comprises silicon-based magnetic beads and 60% -80% alcohol washing liquid, hydroxyl or carboxyl is modified on the surfaces of the silicon-based magnetic beads, the particle size of the magnetic beads is 50-400nm, and the content of the magnetic beads is 1-2mg/mL.

Preferably, the blood RNA extraction kit further comprises a washing solution I, wherein the washing solution I comprises 1-4M guanidine isothiocyanate, 1-50mM EDTA, 10-70% ethanol and 0.5-10% NP-40.

Preferably, the blood RNA extraction kit further comprises one or more of a washing solution II, DNase I and an eluent.

Preferably, the DNase I of the blood RNA extraction kit, including but not limited to, DNase I commercially available from Ebolac, zhuhai Sharp, norvezan, promega, NEB or Takara, is used in an amount of 5-20U/sample.

According to another aspect of the present invention, there is also provided an extraction method for extracting blood RNA by a magnetic bead method, which uses the blood RNA extraction kit according to the present invention for extraction.

Preferably, the method for extracting blood RNA comprises the following steps:

adding 300-500 mu L of lysis binding solution according to 200-300 mu L of blood sample, performing room temperature lysis, adding 600-900 mu L of magnetic bead washing solution after leucocyte lysis to obtain mixed solution containing magnetic bead-nucleic acid, and collecting the magnetic bead-nucleic acid in the mixed solution;

washing with washing solution I and washing solution II, adding 50-100 mu L DNase I reaction mixture, digesting to remove genome DNA, and eluting magnetic beads with eluent to obtain the extracted product RNA.

Preferably, the blood sample of the blood RNA extraction method is frozen blood, and the lysis binding solution comprises 1-4M guanidine hydrochloride, 10-60mM thiourea, 10-60mM sodium citrate, 1-30mM EDTA, isopropanol with the mass concentration of 20% -70%, 0.1-20mM sodium chloride, 10-200mM sodium acetate, hexadecyl sarcosine sodium with the mass concentration of 0.1% -4% and dodecyl sodium sulfonate with the mass concentration of 0.1% -3%.

Preferably, the blood sample is refrigerated blood, and the lysis binding solution comprises 1-4M guanidine hydrochloride, 10-60mM thiourea, 10-60mM sodium citrate, 1-20mM EDTA, isopropanol with a mass concentration of 20% -70%, 0.1-20mM sodium chloride, 10-200mM sodium acetate, hexadecyl sarcosine sodium with a mass concentration of 0.1% -4% and dodecyl sodium sulfonate with a mass concentration of 0.1% -3%.

In general, compared with the prior art, the blood RNA extraction kit provided by the invention has the following beneficial effects:

the blood RNA extraction kit provided by the invention comprises a lysis binding solution and a magnetic bead washing solution, wherein an anionic surfactant (sodium cetyl sarcosinate and sodium dodecyl sulfate) in the lysis binding solution can lyse cells and dissolve cell membranes to release RNA and DNA, and sodium acetate and isopropanol in the lysis binding solution can cooperate to obviously promote RNA precipitation, so that RNA and DNA are separated, and meanwhile, sodium acetate can increase the negative charge density of RNA in the solution, so that RNA is combined with silicon-based magnetic beads with surface modified hydroxyl or carboxyl in a specific manner; in addition, the combined action of guanidine hydrochloride, EDTA, thiourea and guanidine salt can inhibit the activity of RNase, prevent RNA degradation and facilitate the extraction of complete RNA from RNase-enriched blood.

The blood RNA extraction kit provided by the invention can directly extract RNA from a refrigerated or frozen whole blood sample without first lysing erythrocytes and centrifugally separating to precipitate white blood cells and then extracting RNA, has a simple extraction process, does not contain genome DNA and small fragment DNA/RNA in an extracted RNA product, and can extract complete RNA in a high flux.

Drawings

FIG. 1 is the agarose electrophoresis running results of example 2 and comparative examples 1 and 2;

FIG. 2 is the result of agarose electrophoresis running of the kit extract product of example 1 and the kit extract product of comparative example 3;

FIG. 3 is the result of agarose electrophoresis running of the kit extract product of example 1 and the kit extract product of comparative example 4;

FIG. 4 shows the result of agarose electrophoresis running of the extracted products of the RNA extraction methods of example 4 and comparative examples 5 and 6.

Detailed Description

In order to further describe the technical means and effects adopted by the present invention for achieving the intended purpose, the following detailed description will refer to the specific implementation, structure, characteristics and effects according to the present invention with reference to the accompanying drawings and preferred embodiments.

The presence of blood components, particularly hemoglobin and various cytokines and secondary metabolites in the blood, presents challenges for RNA extraction, which is better and more complex than plant samples. These components tend to interact with RNA molecules, resulting in degradation or contamination of the RNA during extraction. And red blood cells in blood do not contain RNA, and only 10% of white blood cells by volume of blood are provided with RNA. Therefore, usually, erythrocytes are first lysed, centrifuged, precipitated and collected to collect leukocytes, and then RNA is extracted, but blood is frozen or stored for too long to cause hemolysis, all cells are broken at this time, and leukocytes cannot be separated, and pure RNA is difficult to extract by the split red method (removing erythrocytes with erythrocyte lysate).

The invention provides a magnetic bead method blood RNA extraction kit, which comprises a lysis binding solution and a magnetic bead washing solution;

the lysis binding solution comprises 1-6M guanidine hydrochloride, 10-100mM thiourea, 10-100mM sodium citrate, 1-50mM EDTA, 10-70% (W/V) isopropanol, 0.1-20mM sodium chloride, 10-300mM sodium acetate, 0.1-5% (W/V) hexadecyl sarcosine sodium, 0.1-5% (W/V) sodium dodecyl sulfonate and is prepared from DEPC water, wherein 'W/V' represents mass concentration;

The invention discovers that 10-300mM sodium acetate and 10% -70% (W/V) isopropanol are added into the lysis binding solution, can obviously promote RNA precipitation and is beneficial to separating RNA from genome DNA. Wherein sodium acetate can increase negative charge density of RNA in the solution, and is favorable for specific binding of RNA and silicon-based magnetic beads with surface modified hydroxyl or carboxyl. In addition, guanidine hydrochloride in the lysis binding solution is a strong inhibitor of nuclease, meanwhile, EDTA can inhibit the activity of RNase, and can prevent RNA degradation, thus being beneficial to extracting complete RNA from RNase-enriched blood; the thiourea is a protein denaturant, and is used together with guanidine salt, so that the cleavage effect can be enhanced, the RNase activity can be inhibited by damaging the protein structure under the combined action, the RNA degradation can be further prevented, the generation of small fragment RNA can be obviously reduced, more complete RNA can be reserved, and the RNA extraction amount can be improved. And can maintain the stability of RNA structure in the salt ion environment formed by sodium chloride and sodium citrate, and sodium citrate can be used as a pH buffer to help maintain the stable pH value of the solution, which is very important for protecting RNA molecules.

The use of surface modified hydroxyl or carboxyl based magnetic beads in the cleavage-binding system allows specific binding of intact RNA, but not genomic DNA and small or degraded fragments of DNA/RNA, allowing extraction of pure intact RNA.

Further, the frozen blood is extracted, and the preferred lysis conjugate comprises a solution comprising 1-4M guanidine hydrochloride, 10-60mM thiourea, 10-60mM sodium citrate, 1-30mM EDTA, 20% -70% (W/V) isopropyl alcohol, 0.1-20mM sodium chloride, 10-200mM sodium acetate, 0.1% -4% (W/V) hexadecyl sarcosine sodium, 0.1% -3% (W/V) sodium dodecyl sulfonate;

the extraction of refrigerated blood, the preferred lysis binding fluid comprises 1-4M guanidine hydrochloride, 10-60mM thiourea, 10-60mM sodium citrate, 1-20mM EDTA, 20% -70% (W/V) isopropyl alcohol, 0.1-20mM sodium chloride, 10-200mM sodium acetate, 0.1% -4% (W/V) hexadecyl sarcosine sodium, 0.1% -3% (W/V) sodium dodecyl sulfonate;

preferably, the magnetic beads are magnetic beads with hydroxyl modified surfaces, the particle size of the magnetic beads is 50-400nm, and the content is 1-2mg/mL.

More preferably, the blood RNA extraction kit further comprises one or more of a washing solution I, a washing solution II, DNase I and an eluent.

The washing liquid I comprises 1-4M guanidine isothiocyanate, 1-50mM EDTA, 10% -70% (W/V) ethanol and 0.5% -10% (W/V) NP-40; preferably, the kit comprises 1-3M guanidine isothiocyanate, 1-30mM EDTA, 20% -70% (W/V) ethanol, and 0.5% -5% (W/V) NP-40.

The washing liquid II comprises 55% -85% ethanol; preferably 60% -80% ethanol.

The DNase I is used in an amount of 5-20U/sample, including, but not limited to, DNase I commercially available from Aibotac, zhuhai Sharp, norvezan, promega, NEB or Takara.

The eluent comprises sterile DEPC water.

The blood RNA extraction kit can be applied to an automatic nucleic acid extractor for high-throughput extraction.

In addition, the invention also provides an extraction method for extracting blood RNA by a magnetic bead method, which adopts the blood RNA extraction kit to extract.

In some embodiments, the method for extracting blood RNA by the magnetic bead method comprises the steps of:

adding 300-500 mu L of lysis binding solution into 200-300 mu L of blood sample, and lysing white blood cells at room temperature;

adding 600-900 mu LL magnetic bead washing liquid into the pyrolysis liquid after pyrolysis to enable magnetic beads to be specifically combined with nucleic acid, obtaining mixed liquid containing magnetic beads-nucleic acid, and collecting the magnetic beads-nucleic acid in the mixed liquid by utilizing magnetic force adsorption;

respectively adding a washing solution I and a washing solution II, and washing to remove residual salt ions and organic reagents;

adding 50-100 mu L of DNase I reaction mixture, and digesting to remove genome DNA;

and finally adding eluent, and eluting the magnetic beads to obtain the extracted product RNA.

In some embodiments, the blood sample is frozen blood and the lysis binding fluid comprises 1-4M guanidine hydrochloride, 10-60mM thiourea, 10-60mM sodium citrate, 1-30mM EDTA, 20% -70% (W/V) isopropyl alcohol, 0.1-20mM sodium chloride, 10-200mM sodium acetate, 0.1% -4% (W/V) hexadecyl sarcosine sodium, 0.1% -3% (W/V) sodium dodecyl sulfonate.

In some embodiments, the blood sample is chilled blood and the lysis binding fluid comprises 1-4M guanidine hydrochloride, 10-60mM thiourea, 10-60mM sodium citrate, 1-20mM EDTA, 20% -70% (W/V) isopropyl alcohol, 0.1-20mM sodium chloride, 10-200mM sodium acetate, 0.1% -4% (W/V) hexadecyl sarcosine sodium, 0.1% -3% (W/V) sodium dodecyl sulfonate.

The following are examples

Example 1 blood RNA extraction kit

The blood RNA extraction kit comprises a lysis binding solution, a magnetic bead washing solution, a washing solution I, a washing solution II, DNase I, a DNase I reaction buffer solution and an eluent, wherein the lysis binding solution is 2M guanidine hydrochloride, 40mM thiourea, 30mM sodium citrate, 10mM EDTA, 50% (W/V) isopropanol, 0.5mM sodium chloride, 10mM sodium acetate, 0.5% (W/V) hexadecyl sodium sarcosinate and 0.8% (W/V) sodium dodecyl sulfonate;

washing solution I was 1M guanidine isothiocyanate, 5mM EDTA, 50% (W/V) ethanol, and 2% (W/V) NP-40;

the washing liquid II is 70% ethanol, the magnetic bead washing liquid comprises magnetic beads and 60% -80% ethanol washing liquid, wherein the magnetic beads are silicon-based magnetic beads, the particle size range is 50-200nm, the content is 1-2mg/mL, and the surfaces of the silicon-based magnetic beads are modified with hydroxyl groups.

DNase I and DNase I buffer DNase I from Promega was used and 5-15U/sample was added according to the instructions.

In the present invention, DNase I and DNase I buffers may be commercially available DNase I from Aibotac, zhuhai Sharp, norvezan, NEB or takara, and may be formulated to be added in an amount of 5-15U/sample according to the specifications of each brand.

EXAMPLE 2 extraction of Total RNA from cryopreserved blood

In this example, the blood sample was blood stored at 4℃for one month, and total RNA was extracted using the RNA extraction kit of example 1 in a NanomagBio S-48 fully automatic nucleic acid extractor, as follows:

taking a blood sample stored at 4 ℃ for one month, and mixing in a biosafety cabinet for later use;

the instrument power was turned on and instrument parameters were set as shown in the following table:

TABLE 1 blood Total RNA extraction procedure

Step (a)

1 step

2 steps

3 steps

4 steps

5 steps

6 steps

7 steps

8 steps

Step 9

Station

4

1

2

3

4

6

5

4

Latency time

/

3min

/

Hybrid mode

2

1

2

1

2

1

2

Mixing time

/

7min

2min

15min

2min

5min

30s

Whether to pause or not

Whether or not

Is that

Whether or not

Magnet attraction time

1min

2min

1min

/

1min

/

Temperature (temperature)

/

25℃

/

55℃

/

Taking out 1 96-well plate, and adding each component reagent into the corresponding hole site of the deep-hole plate according to the following table in advance.

TABLE 2 split charging amount of whole blood RNA extraction reagent and corresponding position of deep-hole plate

Station	Deep hole plate hole	Adding reagent and volume
			1	Column 1/7	300-500 mu L of lysis binding solution
2	Column 2/8	500-1000 mu L of washing liquid I
			3	Column 3/9	500-1000 mu L of washing liquid I
4	Column 4/10	600-900 mu L of washing liquid II+20 mu L of magnetic beads
			5	Column 5/11	35-100μL RNase-free water
6	Column 6/12	100 mu L DNase I reaction mixture

10 mu L of proteinase K and 200-500 mu L of whole blood sample are added into the lysis binding solution of the 1/7 th row hole site of each deep hole plate, and the mixture is gently blown by a gun head for 5-6 times to be uniformly mixed. And (3) placing the deep hole plate into a clamping groove of the instrument, placing a magnetic rod sleeve, closing the cabin door, and starting a running program.

After the program is suspended, the 96-well plate is taken out, 500-1000uL of washing liquid II is added to the 6/12 th column, then the 96-well plate is put back into a clamping groove of the instrument, and the program is continuously operated.

After the procedure was completed, the 96-well plate was removed, and 5/11 column samples were transferred to a clean centrifuge tube of RNase-free, which was the extracted blood RNA product.

Comparative example 1

The blood RNA extraction kit of Zymo brand is adopted, the operation is strictly carried out according to the specification of the kit, and the total blood RNA is extracted as follows:

200. Mu.L of DNA/RNA protectant was added to 200. Mu.L of whole blood sample and mixed well, followed by 8. Mu.L of proteinase K and mixed well. Incubate at room temperature (20-30 ℃) for 30min. An equal volume of isopropanol was then added and vortexed. The well mixed sample was added to column 3G and centrifuged in a collection tube, and column 3G was transferred to a clean RNase-free centrifuge tube. 200. Mu.L of the RNA recovered solution was added to a green column No. 3 and centrifuged. 200. Mu.L of ethanol (95-100%) was then added to the filtrate from the previous step and mixed well.

The mixture was added to a white column No. 1 and placed in a collection tube, centrifuged, and the filtrate was decanted. 400. Mu.L of RNA washing solution was added to column C, and the mixture was centrifuged to remove the filtrate. For each sample treatment, 40. Mu.L of DNase I reaction solution was prepared. 35. Mu.L of DNase I digestion solution was added directly to 40. Mu.L of DNase I reaction solution on column 1 and incubated at room temperature (20-30 ℃) for 15 minutes.

After the digestion treatment, 400. Mu.l of RNA pre-wash was added to column 1, centrifuged and the filtrate removed. After that, the column 1 was washed twice with RNA washing solution, centrifuged, and the filtrate was removed. Idle for 2 minutes to remove residual ethanol so as not to inhibit downstream reactions. Finally, the column 1 was removed, placed in a centrifuge tube without RNase, 15. Mu.L of RNase-free water was added to the middle portion of the adsorption membrane, left at room temperature for 2 minutes, and centrifuged for 1 minute to elute RNA.

Comparative example 2

The preparation method comprises the following steps of strictly executing operation according to a reagent kit instruction by adopting a blood RNA extraction reagent kit of a Kanji brand, extracting total RNA of precipitated white blood cells after erythrocyte lysis, and specifically comprising the following steps:

firstly, according to 1: 5. Buffer EL was added to the blood and mixed well, and incubated on ice for 10-15 minutes. During incubation, mix 2 times by brief vortexing. The cloudy suspension became translucent during incubation, indicating erythrocyte lysis. If necessary, the incubation time can be extended to 20 minutes. Centrifuge at 4 ℃,400x g for 10 minutes, completely remove and discard the supernatant.

Buffer EL was added to the cell pellet at 2 times the whole blood volume. Cells were resuspended by brief vortexing. Buffer RLT was added to granular white blood cells and vortexed or pipetted as indicated in the following table.

TABLE 3Buffer RLT addition

Buffer RLT(μl)	Whole blood (ml)	Leukocyte count
			350	Up to 0.5	Up to 2×10 ⁶
600	0.5to 1.5	2×10 ⁶ to 1×10 ⁷

The lysate was transferred directly into a QIAshredder column and packed in a 2ml collection tube and centrifuged at maximum speed for 2min. The QIAshredder column was discarded, 1 volume (350. Mu.L or 600. Mu.L) of 70% ethanol was added to the homogeneous lysate and mixed by pipetting. Without centrifugation, a precipitate forms upon addition of ethanol. The sample was carefully transferred to a new QIAamp column and placed in a 2mL collection tube and centrifuged at 8000x g for 15s. The QIAamp column was placed in a new 2mL collection tube. Add 500. Mu.l Buffer RPE, > 8000x g (> 10,000 rpm) to the column and centrifuge for 15s, discard the filtrate and collection tube.

The QIAamp column was carefully opened and 500. Mu.l Buffer RPE was added. The lid was closed and centrifuged at full speed (20,000Xg, 14,000 rpm) for 3min followed by idle centrifugation for 1min. Placing the adsorption column into a 1.5ml microcentrifuge tube, adding 30-50 μl of RNase-free water, centrifuging at 8000x g (more than or equal to 10000 rpm) for 1min, and eluting to obtain RNA extract product.

The quality of the blood RNA products extracted in example 2, comparative example 1 and comparative example 2 was examined, and the absorbance values of the RNA products at the spectrophotometric absorption peaks 230nm,260nm and A280nm were specifically examined, and the results are shown in Table 4, and the RNA extracted products were subjected to 1.2% ordinary agarose gel electrophoresis, and the electrophoresis results are shown in FIG. 1.

TABLE 4 spectrophotometric measurement of total RNA products of blood extracted from different kits

As shown in Table 4, the total RNA extraction kit for blood provided by the invention is matched with a NanoMagBio S-48 full-automatic nucleic acid extractor, a blood sample stored for one month at 4 ℃ is extracted, 3 samples are repeatedly detected, the concentration of the extracted total RNA product is highest, the volume of the actual eluted product is about 30 mu L, the average yield is about 3 mu g, the A260/A280 ratio is basically about 2.0, the A260/A230 ratio is about 1.97, and the extraction purity is high.

The elution volumes of RNA products extracted by the Zymo brand kit and the Kanji brand kit are 30 mu L, the average yields are 1.37 mu g and 0.64 mu g respectively, and the RNA yields extracted by the kit are 2.18 times and 4.68 times that of the Zymo brand kit and the Kanji brand kit respectively. Therefore, the blood RNA extraction kit provided by the invention can obviously improve the yield of the extracted RNA product.

In FIG. 1, the Marker used was a foolish DL2000 DNA Marker, wherein the Marker loading was 1. Mu.L, the extract loading was 10. Mu.L, the Zymo was labeled as the result of running the extract of the Zymo brand blood RNA kit, the Kaiji was labeled as the result of running the extract of the Kaiji brand blood RNA kit, and the nanomagnetic self-polishing was labeled as the result of running the extract of the kit of the present invention.

As can be seen from FIG. 1, the common gel electrophoresis pattern of the RNA products extracted by the three blood RNA kits provided by the invention only clearly shows 28SRNA and 18S RNA bands in the products extracted by the blood RNA extraction kit provided by the invention, more genome DNA (gDNA) is mixed in the RNA extracted by adopting the Kaijer brand blood RNA kit, and small fragment DNA/RNA remains in the RNA extracted by adopting the Zymo brand blood RNA kit, which indicates that the RNA extracted by adopting the blood RNA extraction kit provided by the invention has complete structure and no small fragment RNA and DNA residues. Therefore, the blood RNA extraction kit provided by the invention not only can extract high-concentration and high-purity blood total RNA, but also has complete extracted RNA structure.

Compared with the commercial Zymo brand and Kaiji brand kits, the blood total RNA extracted by the kit has no small fragment RNA, the genome DNA (gDNA) is removed cleanly, no residue is left, the magnetic bead method is utilized to match with a full-automatic nucleic acid extractor, the whole extraction process is simple and short in operation time, extremely high in automation degree and low in cost, and the kit is particularly suitable for high-throughput automatic blood total RNA extraction analysis.

Comparative example 3

The lysis binding solution comprises 3M guanidine hydrochloride, 40mM thiourea, 30M sodium citrate, 7.5mM EDTA, 100mM sodium acetate, 0.5mM sodium chloride, 2% (W/V) hexadecyl sarcosinate, 0.8% (W/V) sodium dodecyl sulfonate;

the washing solution I, the bead washing solution, the washing solution II, the DNase I reaction solution and the eluent were the same as those in example 1.

The extraction procedure was as in example 1. The quality of the total RNA product of the extracted refrigerated blood sample is detected, and the absorbance values of the RNA product at the spectrophotometric absorption peaks A230nm, A260nm and A280nm are specifically detected, as shown in Table 5.

TABLE 5 spectrophotometric measurement of total RNA product from frozen blood samples

Blood sample	RNA(ng/μL)	A260/A280	A260/A230
				1	101.21	2.105	1.726
2	105.247	2.062	1.745
				3	110.46	2.034	1.832

As is clear from Table 5, the blood RNA extraction kit of comparative example 3 was used to extract chilled blood RNA, the total RNA product concentration was 105.64 ng/. Mu.L on average, the actual elution volume was about 30. Mu.L, the total yield was about 3. Mu.g, the A260/A280 ratio was substantially 2.0 or more, and the A260/A230 ratio was about 1.75.

The results of 1.2% ordinary agarose gel electrophoresis of the blood RNA extracted using the kit of example 1 and the RNA extracted from the chilled blood sample extracted in comparative example 3 are shown in FIG. 2. In FIG. 2, the Marker was used as a fool DL2000 DNA Marker, wherein the Marker loading was 1. Mu.L and the extract loading was 10. Mu.L.

As is clear from FIG. 2, the gDNA residue was found on the 28S RNA of the extracted product of comparative example 3, which indicates that genomic DNA was found in the extracted blood RNA, whereas no genomic DNA was found in the blood RNA extracted by the blood RNA extraction kit provided by the present invention.

Comparative example 4

The lysis binding solution comprises 3M guanidine hydrochloride, 40mM thiourea, 50M sodium citrate, 10mM EDTA, 10mM sodium chloride, 45% (W/V) isopropanol, 2% (W/V) hexadecyl sarcosine sodium, 1% (W/V) sodium dodecyl sulfonate;

washing solution I comprises 1M guanidine isothiocyanate, 50% (W/V) ethanol and 2% (W/V) NP-40; the bead washing solution, washing solution II, DNase I reaction solution and eluent were the same as those in example 1.

The extraction procedure was as in example 1. The quality of the total RNA product of the extracted refrigerated blood sample is detected, and the absorbance values of the RNA product at the spectrophotometric absorption peaks A230nm, A260nm and A280nm are specifically detected as shown in Table 6.

TABLE 6 Spectrophotometer measurement results of total RNA products of extracted frozen blood samples

Blood sample	RNA(ng/μL)	A260/A280	A260/A230
				1	85.262	2.105	1.738
2	84.347	2.062	1.846
				3	82.463	2.034	1.795

The RNA extraction kit of comparative example 4 was used to extract total RNA from chilled blood, the total RNA product concentration was on average 84.024 ng/. Mu.L, the actual elution volume was about 30. Mu.L, the total RNA extraction was about 2.5. Mu.g, the A260/A280 ratio was substantially above 2.0, and the A260/A230 ratio was about 1.8.

The blood RNA extracted from the kit of example 1 and the RNA extracted from the chilled blood sample extracted from comparative example 4 were subjected to 1.2% ordinary agarose gel electrophoresis, and the results are shown in FIG. 3. In FIG. 3, a Marker was used for the fool DL2000 DNA Marker, wherein the Marker loading was 1. Mu.L and the extract loading was 10. Mu.L.

As can be seen from FIG. 3, in comparative example 4, there is a small fragment DNA/RNA residue below 18S RNA, which is the RNA extracted from the blood sample, whereas the present invention provides a kit for extracting blood RNA without a small fragment DNA/RNA residue.

Example 3 blood RNA extraction kit

The blood RNA extraction kit comprises a lysis binding solution, a magnetic bead washing solution, a washing solution I, a washing solution II, DNase I, a DNase I reaction buffer solution and an eluent, wherein the lysis binding solution is 2.5M guanidine hydrochloride, 50mM thiourea, 20mM sodium citrate, 5mM EDTA, 20% (W/V) isopropanol, 0.5mM sodium chloride, 100mM sodium acetate, 3% (W/V) hexadecyl sarcosine sodium and 1% (W/V) dodecyl sodium sulfonate;

washing solution I was 3M guanidine isothiocyanate, 10mM EDTA, 45% (W/V) ethanol, and 2% (W/V) NP-40;

the washing liquid II is 70% ethanol, the magnetic bead washing liquid comprises magnetic beads and 60% -80% ethanol washing liquid, wherein the magnetic beads are silicon-based magnetic beads, the particle size range is 50-200nm, the content is 1-2mg/mL, and carboxyl groups are modified on the surfaces of the silicon-based magnetic beads.

DNase I and DNase I buffer were DNase I supplied by NEB and were added in 5-20U/sample according to the instructions of each brand.

EXAMPLE 4 extraction of frozen blood Total RNA

In this example, the blood sample was blood stored at-80℃for one month, and total blood RNA was extracted using the blood RNA extraction kit of example 3 in a NanomagBio S-48 full-automatic nucleic acid extractor, as follows:

taking a blood sample frozen at the temperature of minus 80 ℃ and storing the blood sample in a biosafety cabinet for later use;

turning on the power of the instrument, and setting instrument parameters according to the table 7;

TABLE 7 blood Total RNA extraction procedure

1 96-well plate is taken out, and each component reagent is added into the corresponding hole sites of the deep-hole plate in turn according to the table 8 in advance.

TABLE 8 split charging amount of blood RNA extraction reagent and corresponding position of deep-hole plate

Station	Deep hole plate hole array	Adding reagent and volume
			1	Column 1/7	500 mu L of lysis conjugate
2	Column 2/8	800 mu L of washing liquid I
			3	Column 3/9	800 mu L of washing liquid I
4	Column 4/10	900 mu L of washing liquid II+20 mu L of magnetic beads
			5	Column 5/11	35-100μL RNase-free water
6	Column 6/12	100 mu L DNase I reaction mixture

10 mu L of proteinase K and 200-500 mu L of whole blood sample are added into the lysis binding solution of the 1/7 th row hole site of each deep hole plate, and the mixture is gently blown by a gun head for 5-6 times to be uniformly mixed.

And (3) placing the deep hole plate into a clamping groove of the instrument, placing a magnetic rod sleeve, closing the cabin door, and starting a running program.

After the program is suspended, the 96-well plate is taken out, 500-1000 mu L of washing liquid II is added to the 6/12 th column, then the 96-well plate is put back into a clamping groove of the instrument, and the program is continuously operated.

Comparative example 5

Blood RNA was extracted by Trizol LS method using the frozen blood of example 4 as the extraction target, and the specific procedures were strictly performed according to the reagent specifications as follows:

a250. Mu.L blood sample was taken and added to a 1.5mL EP tube containing 750. Mu.L Trizol-LS. Immediately after Trizol-LS is added, the mixture is inverted and mixed evenly, and the mixture is stood for 5min at room temperature, so that the nucleic acid protein complex is completely separated. 200. Mu.L of chloroform was then added thereto, and the mixture was vigorously shaken for 15 seconds and left at room temperature for 2 minutes. Centrifuge at 13000rpm for 10min and aspirate 600. Mu.L of colorless supernatant into a new 1.5mL EP tube.

600. Mu.L of isopropyl alcohol was added to the 600. Mu.L supernatant, and the mixture was turned upside down with force several times and left at-20℃for 5 minutes. Centrifugation at 13000rpm for 10min, careful decanting of the supernatant, leaving a bottom total RNA pellet. 1mL of 70% ethanol was added to each tube of the pellet, and the pellet was centrifuged at 13,000rpm for 5min, and the supernatant was carefully decanted. The washing was repeated once more, the washing was decanted, centrifuged again for 10 seconds, and after the remaining washing was blotted with a 10. Mu.L Tip, the mixture was left at room temperature to evaporate the ethanol (20 min). Adding 20-100 mu L TE Buffer or RNase Free H into each tube ₂ O solubilizes total RNA.

Comparative example 6

Blood RNA was extracted by Trizol method using the frozen blood of example 4 as the extraction target, and the specific procedures were strictly carried out according to the reagent specifications as follows:

a250. Mu.L blood sample was taken and added to a 1.5mL EP tube containing 750. Mu.L Trizol. Immediately after Trizol is added, the mixture is inverted and mixed evenly, and the mixture is stood for 5min at room temperature, so that the nucleic acid protein complex is completely separated. 200. Mu.L of chloroform was then added thereto, and the mixture was vigorously shaken for 15 seconds and left at room temperature for 2 minutes. Centrifuge at 13,000rpm for 10min and aspirate 600. Mu.L of colorless supernatant into a fresh 1.5mL EP tube.

600. Mu.L of isopropyl alcohol was added to the 600. Mu.L supernatant, and the mixture was turned upside down with force several times and left at-20℃for 5 minutes. Centrifugation at 13,000rpm for 10min, carefully decanting the supernatant, leaving a bottom total RNA pellet. 1mL of 70% ethanol was added to each tube of the pellet, and the pellet was centrifuged at 13,000rpm for 5min, and the supernatant was carefully decanted. The washing was repeated once more, the washing was decanted, centrifuged again for 10 seconds, and after 10. Mu.l Tip was used to blot the remaining washing, the mixture was left at room temperature to evaporate the ethanol (20 min). 20-100 mu L TE Buffer or RNase Free H2O is added into each tube to dissolve total RNA.

The quality of the blood RNA products extracted in example 4, comparative example 5 and comparative example 6 was examined, and the absorbance values of the RNA products at the spectrophotometric absorption peaks 230nm,260nm and A280nm were specifically examined, and the results are shown in Table 9, and the RNA extracted products were subjected to 1.2% ordinary agarose gel electrophoresis, and the electrophoresis results are shown in FIG. 4.

TABLE 9 spectrophotometric measurements of total RNA products from blood extracted by different methods

As shown in Table 9, the blood RNA extraction reagent provided by the invention is used for extracting blood samples at the temperature of minus 80 ℃,3 samples are repeatedly detected, the total concentration of extracted RNA is 69.8 ng/. Mu.L, the actual elution volume is about 50. Mu.L, the calculated extraction amount of the obtained RNA product is about 3.5. Mu.g, the ratio of A260/A280 is basically more than 1.9, and the ratio of A260/A230 is about 1.7.

While the elution volumes of the RNA products extracted by Trizol LS and Trizol were 50. Mu.L, and the average yields were 2.5. Mu.g and 3.5. Mu.g, respectively, the RNA yield was virtually high because of the large amount of genomic DNA remaining in the Trizol extract.

In FIG. 4, a Marker was used as a fool FY5000 DNA Marker (EG 21911M), the Marker loading amount was 1. Mu.L, trizol LS was labeled as the result of running the gel of the extract product of Trizol LS for extracting blood RNA, trizol was labeled as the result of running the gel of the extract product of blood RNA using Trizol, and nanomagnetic self-grinding was labeled as the result of running the gel of the extract product of the kit of example 3.

As can be seen from FIG. 4, the common gel electrophoresis patterns of the three extraction methods can show clear 28S and 18S bands, but blood RNA extracted by Trizol LS and Trizol method is mixed with small fragment RNA, blood RNA extracted by Trizol method is mixed with genome DNA, and RNA extracted by the blood RNA extraction kit provided by the invention does not contain genome DNA and small fragment DNA/RNA, and the extracted RNA has high purity and complete structure.

The present invention is not limited to the above embodiments, but is capable of modification and variation in detail, and other modifications and variations can be made by those skilled in the art without departing from the scope of the present invention.

Claims

1. An extraction kit for extracting blood RNA by a magnetic bead method is characterized by comprising a lysis binding solution and a magnetic bead washing solution;

2. The kit for extracting blood RNA by the magnetic bead method according to claim 1, wherein the frozen or chilled blood is extracted, and the lysis binding solution comprises 1-5M guanidine hydrochloride, 10-80mM thiourea, 10-80mM sodium citrate, 1-40mM EDTA, isopropanol with a mass concentration of 20% -70%, 0.1-20mM sodium chloride, 10-300mM sodium acetate, hexadecyl sodium sarcosinate with a mass concentration of 0.1% -4% and sodium dodecyl sulfonate with a mass concentration of 0.1% -4%.

3. The kit for extracting blood RNA by a magnetic bead method according to claim 1 or 2, wherein the magnetic bead washing liquid comprises silicon-based magnetic beads and 60% -80% alcohol washing liquid, the surfaces of the silicon-based magnetic beads are modified with hydroxyl or carboxyl, the particle size of the magnetic beads is 50-400nm, and the content of the magnetic beads is 1-2mg/mL.

4. The blood RNA extraction kit of any one of claims 1 to 3, further comprising a wash solution i comprising 1-4M guanidine isothiocyanate, 1-50mM EDTA, 10% -70% ethanol by mass and 0.5% -10% NP-40 by mass.

5. The kit for extracting blood RNA as claimed in claim 4, further comprising one or more of washing solution II, DNase I and eluent _。

6. The blood RNA extraction kit of claim 5, wherein DNase I is used in an amount of 5-20U/sample, including, but not limited to, DNase I commercially available from Aibotac, zhuhai, norvezan, promega, NEB or Takara.

7. An extraction method for extracting blood RNA by a magnetic bead method, which is characterized in that the blood RNA extraction kit according to any one of claims 1 to 6 is used for extraction.

8. The method for extracting blood RNA as defined in claim 7, comprising the steps of:

9. The method for extracting blood RNA according to claim 8, wherein the blood sample is frozen blood, and the lysis binding solution comprises 1-4M guanidine hydrochloride, 10-60mM thiourea, 10-60mM sodium citrate, 1-30mM EDTA, isopropanol with a mass concentration of 20% -70%, 0.1-20mM sodium chloride, 10-200mM sodium acetate, hexadecyl sodium sarcosinate with a mass concentration of 0.1% -4%, and sodium dodecyl sulfonate with a mass concentration of 0.1% -3%.

10. The method for extracting blood RNA according to claim 8, wherein the blood sample is chilled blood, and the lysis binding solution comprises 1-4M guanidine hydrochloride, 10-60mM thiourea, 10-60mM sodium citrate, 1-20mM EDTA, isopropanol with a mass concentration of 20% -70%, 0.1-20mM sodium chloride, 10-200mM sodium acetate, hexadecyl sodium sarcosinate with a mass concentration of 0.1% -4%, and sodium dodecyl sulfonate with a mass concentration of 0.1% -3%.