CN114891784B - Extraction method of semen RNA - Google Patents
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- CN114891784B CN114891784B CN202210823067.XA CN202210823067A CN114891784B CN 114891784 B CN114891784 B CN 114891784B CN 202210823067 A CN202210823067 A CN 202210823067A CN 114891784 B CN114891784 B CN 114891784B
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Abstract
The invention provides a semen RNA extraction method, which comprises the steps of resuspending a sperm precipitate obtained after semen pretreatment in PBS, processing the sperm precipitate overnight by an RNAscope, centrifuging to obtain a sperm precipitate, adding RL lysate into the sperm precipitate, carrying out water bath at 60 ℃ until the solution is transparent, processing the sperm precipitate by a TRIzol reagent, centrifuging to obtain a supernatant, and recovering RNA by a centrifugal column method. The concentration of RNA extracted by the method is obviously higher than that of the RNA extracted by the conventional RNA extraction method, and the integrity and purity of the RNA are high. The method has good application prospect.
Description
Technical Field
The invention relates to the field of nucleic acid extraction, in particular to a method for extracting semen RNA.
Background
The sperm is a highly differentiated haploid cell which is formed by the spermatogonial stem cell through the processes of mitosis, meiosis, deformation and the like and is characterized by chromatin condensation and organelle discharge. Researches find that the RNA content of the sperms of the mammals is very low, and the difficulty of extracting complete RNA is high. In addition, a large number of disulfide bonds exist on the surface of the cell membrane of the cow sperm to maintain the stability of the membrane structure, and the disulfide bonds cannot be broken by common reagents, so that the extraction of the cow sperm RNA and protein is difficult.
The current common RNA extraction methods include TRIzol method and centrifugal column method. The TRIzol method is typically a TRIzol commercial kit from Invitrogen, and the spin column method is typically a RNA/DNA/protein extraction kit from Tiangen. When the inventors used these two typical commercial kits to extract bovine sperm RNA according to the instructions, the extraction efficiency was not high.
Chen et al added 5% beta mercaptoethanol to TRIzol, and water bath at 60 deg.C for 30min to completely lyse bovine sperm and obtain RNA with high concentration. But the obtained RNA amount still has a great promotion space.
Disclosure of Invention
The invention aims to provide a semen RAN extraction method aiming at the defects in semen RNA extraction in the prior art.
In order to achieve the purpose, the technical scheme of the invention is as follows:
a semen RNA extraction method comprises the following steps:
1) Adding the semen into an RNase-Free centrifuge tube, centrifuging to obtain sperm precipitate, resuspending the sperm precipitate in PBS, treating with RNAscope at 4 ℃ overnight, adding 2 times volume of precooled PBS, centrifuging at 4 ℃ by 5000 Xg for 5min, discarding the supernatant, and placing the sperm precipitate on ice for later use;
2) Adding RL lysate into the sperm precipitate, performing water bath at 60 ℃ for 3min until the solution is transparent, adding a TRIzol reagent with the same volume as the RL lysate into a centrifuge tube, performing vortex for 15s, standing for 3min, performing centrifugation at 4 ℃ at 15000 Xg for 15min, and taking the supernatant;
3) Recovering the RNA.
Further, the method for recovering RNA in the step 3) is as follows: adding equal volume of absolute ethyl alcohol into the supernate obtained in the step 2), gently and uniformly mixing, transferring into an RNase-Free adsorption column CR3 for adsorption, centrifuging to remove waste liquid, transferring the RNase-Free adsorption column CR3 into a collecting tube, adding deproteinizing liquid into the RNase-Free adsorption column CR3, centrifuging to remove waste liquid, adding rinsing liquid into the RNase-Free adsorption column CR3, standing at room temperature for 2min, centrifuging to remove waste liquid, repeatedly rinsing once, standing and drying, transferring the RNase-Free adsorption column CR3 into a new RNase-Free centrifuge tube, adding RNase-Free ddH2O, standing at room temperature for 2min, and centrifuging to obtain an RNA solution.
Further, the amount of the lysis solution in the step 2) is 500. Mu.L.
Further, the method for recovering RNA in the step 3) is as follows: adding equal volume of absolute ethyl alcohol into 500 mu L of the supernatant obtained in the step 2), gently mixing uniformly, transferring into an RNase-Free adsorption column CR3 for adsorption, centrifuging 30-60S to remove waste liquid by 13,400 Xg, transferring the RNase-Free adsorption column CR3 into a collecting tube, adding 700 mu L of deproteinizing liquid into the RNase-Free adsorption column CR3, centrifuging 30-60S to remove waste liquid by 13,400 Xg, adding rinsing liquid into the RNase-Free adsorption column CR3, standing at room temperature for 2min,13,400 Xg to centrifuge 30-60S to remove waste liquid, repeatedly rinsing once, standing for air drying, transferring the RNase-Free adsorption column CR3 into a new RNase-Free centrifuge tube, adding RNase-Free ddH2O, standing at room temperature for 2min,13,400 Xg to centrifuge 2min to obtain an RNA solution.
In the present invention, the semen may be bovine, equine, porcine, ovine, canine or human semen, and in the embodiments of the present invention, the semen is bovine semen.
The RL lysate, the RNase-Free adsorption column CR3, the deproteinizing liquid and the rinsing liquid in the invention are reagent materials in a TIANGEN RNA extraction kit (the product number is DP 423).
The invention relates to a method for extracting a plurality of RNAs from cow sperms, which comprises 4 methods of extracting RNAs by using TRIzol, thermal TRIzol and TIANGEN RNA extraction kits and a TIANGEN RNA extraction kit + TRIzol method. The concentration of RNA extracted by the TIANGEN RNA extraction kit and the TRIZOL method is obviously higher than that of the TRIZOL and TIANGEN RNA extraction reagent box group (P < 0.05), the OD value is obviously higher than that of the TRIZOL group and the thermal TRIZOL group (P < 0.05), but the difference with the TIANGEN RNA extraction reagent box group is not obvious (P > 0.05), and the RIN value of the TIANGEN RNA extraction kit and the TRIZOL group is obviously higher than that of the other three groups (P < 0.05). The TIANGEN RNA extraction kit and the TRIzol method can effectively extract RNA in sperms.
Drawings
FIG. 1 is a graph showing the effect of different lysates on sperm lysis levels, wherein ABCDE is a group of TRIzol, a group of thermal TRIzol, a group of TIANGEN reagent kit, a group of TIANGEN kit + TRIzol (1), and a group of TIANGEN kit + TRIzol (2);
FIG. 2 shows RNA agarose gel electrophoresis.
Detailed Description
The following examples are intended to further illustrate the present invention but should not be construed as limiting the invention and modifications or wetting of the technical aspects of the present invention are within the scope of the present invention without departing from the spirit and nature of the present invention.
Example 1
1.1 materials and methods
1.1.1 materials
Taking 10 sex-controlled frozen sperms of 3 cows respectively, extracting RNA, and repeating each group of experiments for 6 times.
1.1.2 Main instrumentation
Name of the manufacturer
Shanghai department assistant for 37 ℃ constant-temperature water bath pot
Clear and magnificent same prescription of sperm (microorganism) dynamic and static analysis system
Gun head Axygen
Centrifuge tube Axygen
1mL syringe golden tower
Enzyme-linked immunosorbent assay (TICAN)
BIO-RAD of ordinary PCR instrument
200 microliter centrifuge tube Axygen
BIO-RAD electrophoresis apparatus
Gel imaging System BIO-RAD
BIO-RAD (polymerase chain reaction) instrument of fluorescent quantitative PCR (polymerase chain reaction)
Protein transfer system BIO-RAD
50 mu L centrifuge tube Axygen
Refrigerator Haier at 4 DEG C
Refrigerator Haier at-80 deg.C
1.1.3 preparation of Primary reagents and solutions
Name of the manufacturer
TRIzol (cat # 15596026) Invitrogen
RNA/DNA/protein extraction kit (cat # DP 423) TIANGEN
DNAse 1 TIANGEN
Tianjin Zhiyuan chloroform
Isopropyl alcohol AMRESCO
Tianjin Zhiyuan alcohol
DEPC Water MACGENE
RNA-free water TIANGEN
Beta-mercaptoethanol SIGMA
PBS HYCLONE
RNA store TIANGEN
Glycogen Biyuntian
Agarose BIOWEST
Agarose nucleic acid dye TIANGEN
DNA loading buffer TIANGEN
DNA ladder marker TIANGEN
Reverse transcription kit BIO-RAD
SYRB enzyme BIO-RAD
50XTAB Biyuntian
Biyunyan (RiPA) lysate
4XLoading buffer BIO-RAD
SDS SIGMA
Pepsin inhibitor Biyuntian
PMSF Biyun sky
Byunyan day BCA protein concentration kit
Bio-RAD 10% premix stain free gum BIO-RAD
Bio-RAD 12% premix stain free rubber BIO-RAD
TEMED SIGMA
Prestaining protein marker Thermo Fisher
Non-prestained protein marker BIO-RAD
Biyuntian electrophoresis liquid
Semi-dry filming liquid Biyuntian
PVDF film Biyuntian
TBST MACGENE
Closed liquid Biyuntian
Skimmed milk BIO-RAD
ECL color developing solution BIO-RAD
EDTA SIGMA
1.2 test methods
1.2.1 semen thawing
Quickly taking out the sex-controlled frozen semen from liquid nitrogen by using a forceps, placing the sex-controlled frozen semen in a 37 ℃ constant-temperature water bath kettle to shake and thaw for 30s, wiping a thin tube by using absorbent paper after thawing, collecting the semen in a 1.5mL centrifuge tube, centrifuging for 5min at room temperature of 378 Xg, discarding the supernatant, adding 1mL PBS again at 37 ℃ to resuspend the semen, centrifuging for 5min at room temperature of 378 Xg, discarding the supernatant, and repeating twice.
1.2.2 sperm RNA extraction
The centrifuge tubes were placed on ice. To the sperm pellet was added 300. Mu.L of RNAscope, and the pellet was pipetted evenly and placed in a refrigerator at 4 ℃ overnight. Taking the sperm out of the refrigerator and placing on ice, adding 2 times volume of precooled PBS, centrifuging at 4 ℃ for 5min at 5000 g, removing supernatant, and placing the sperm precipitate on ice for later use.
1.2.2.1 Normal TRIzol method
Operate according to TRIzol reagent Specification
1.2.2.2 thermal TRIzol method
According to the following
The extraction method comprises adding 1ml of Trizol reagent and 50 mu L of beta-mercaptoethanol into sperm precipitate, vortex and shake for 30s until the sperm precipitate is uniformly distributed in the TRIzol reagent, placing the mixed solution in water bath at 60 ℃ for 30min until the solution is transparent, observing under a microscope, placing 10 mu L of the mixed solution on a glass slideAnd (4) covering a cover for microscopic examination to ensure that the sperm head is completely lysed and has no complete sperm morphology. Taking out and placing on ice for total RNA extraction. To 1mL of TRIzol lysate, 200. Mu.L of chloroform was added, vigorously shaken for 15 seconds, and allowed to stand for 5min. Centrifuge at 15000 Xg for 15min at 4 ℃ and carefully aspirate the supernatant 400. Mu.L. Adding 1 volume of isopropanol and 1 μ L of nucleic acid precipitation aid, inverting for several times, standing for 10min, and performing other steps according to the instructions of Trizol reagent.
1.2.2.3 day root RNA kit method
Following the instructions of the TIANGEN RNA extraction kit.
Day 1.2.2.4 RNA kit + TRIzol method (1)
And adding 500 mu L of RL lysate (adding beta-mercaptoethanol into the RL lysate to a final concentration of 1% according to the kit) into the sperm precipitate, carrying out water bath at 60 ℃ for 3min until the solution is transparent, and observing under a microscope until no complete sperm morphology exists. Adding 500 microliters of TRIzol, whirling for 15 seconds, standing for 3min, centrifuging at the temperature of 4 ℃ by 15000 Xg for 15min, taking 500 mu L of supernatant, adding 100% absolute ethyl alcohol by one time volume, and gently mixing. Adsorbing in RNase-Free adsorption column CR3, centrifuging 45S to remove waste liquid by 13,400 Xg, transferring RNase-Free adsorption column CR3 into a collecting tube, adding 700. Mu.L deproteinizing solution into RNase-Free adsorption column CR3, centrifuging 45S to remove waste liquid by 13,400 Xg, adding 500. Mu.L rinsing solution into RNase-Free adsorption column CR3, standing at room temperature for 2min, centrifuging 45S to remove waste liquid by 13,400 Xg, repeatedly rinsing once, standing, air drying, transferring RNase-Free adsorption column CR3 into a new RNase-Free centrifuge tube, adding RNase-Free ddH 2 O is placed at room temperature for 2min, and centrifuged at 13,400 Xg for 2min to obtain an RNA solution.
1.2.2.5 day root RNA kit + TRIzol method (2)
The procedure was as in 1.2.2.4, but no additional beta-mercaptoethanol was added to the lysate.
1.3 statistical methods
Data were analyzed and compared for significance of differences using one-way analysis of variance with the SPSS 25.0 statistical software and SNK multiple comparisons. The results are expressed as Mean ± standard deviation (Mean ± SD),P<0.05 It is indicated that the difference is significant and,P>0.05 means no significant difference.
2. As a result, the
2.1 comparison of sperm lysis by different reagents
From the morphology of cow sperm, no intact sperm morphology was found in the three lysates except for TRIzol group, as shown in fig. 1. In addition to the TRIzol group, the other three groups were able to rapidly lyse sperm.
2.2 Effect of different methods on RNA concentration, purity and integrity
Among the four different RNA extraction methods, the concentration of RNA extracted by the TIANGEN kit and the TRIzol method is obviously higher than that of other groups (in the RNA concentration level)P<0.05 And the difference between the thermal TRIzol method and the TIANGEN kit method is significant: (P<0.05). On the OD value level, the TIANGEN kit method has no significant difference from the TIANGEN kit + TRIzol method（P>0.05 But both were significantly higher than TRIzol and thermal TRIzol(s) (ii)P<0.05). At the RIN level, the TIANGEN kit + TRIzol group was significantly higher than the other groups: (P<0.05 As shown in table 2-1. As can be seen, the TIANGEN kit + TRIzol group extracted RNA with the highest concentration, the best OD value and integrity. Therefore, the TIANGEN kit + TRIzol method is adopted as a subsequent RNA extraction method.
TABLE 1 Effect of different extraction methods on sperm RNA quality
Note: in the same column, the different letters are significantly different in representation: (P<0.05 No significant difference in representation between the same letters (a)P >0.05 The same applies below.
2.3 pair of TIANGEN kit
Quality detection of RNA extracted by the method
In RNA nucleic acid electrophoresis, 28s and 18s bands do not exist in the milk cow sperm RNA, which indicates TIANGEN kit +
The RNA extracted by the method has no genomic DNA and somatic cell contamination, as shown in figure 2.
60% -80% of RNAs in the sperm exist in the cell nucleus, and the complete lysis of the sperm head is important for extracting the RNA. Because bovine sperm cell membranes and chromatin have strong sulfide bonds, the normal TRIzo1 method cannot dissolve the cell membranes, so that RNA cannot be timely and effectively released, and the risk of degradation by endogenous RNA enzymes exists. Beta-mercaptoethanol can reduce disulfide bonds to single bond form. It was found that the addition of 5% beta-mercaptoethanol to TRIzo1 rapidly dissolves the sperm membrane, releases RNA, and inhibits rnase activity. 60 ℃ can accelerate lysis of sperm cell membranes, but 60 ℃ is also the optimal temperature for rnase activity, and there is a risk that RNA will be degraded by endogenous rnases during sperm lysis. Therefore, in order to ensure that RNA in the sperms is not degraded in the water bath environment at 60 ℃, the Tiangen RNAscope reagent is adopted to inactivate endogenous RNA enzyme and inhibit RNA degradation.
The RNA content in sperm is influenced by species differences and extraction methods. In human sperm, an average of 15-20fg RNA per sperm, 5-8.4fg RNA per pig sperm, and 12-20fg RNA per sperm in goat sperm were found. Among bovine sperm, wang Yanfeng et al measured that each of the bovine sperm contained 35-56fg RNA using the thermal TRIzol method and the kit method. In the invention, the RNA extraction amount of the TIANGEN kit and the TRIzol method reaches 100-140 fg/sperm.
The concentration of RNA is only a measure of RNA, and the purity and integrity of RNA are crucial to downstream experiments. Organic reagents, proteins, genomic residues, and the like can all affect the accuracy of downstream experiments. OD values of 1.9-2.1 indicated no contamination of RNA. In the experiment, the OD value of the heat TRIzol for extracting the bovine sperm RNA is between 1.65 and 1.77, which shows that the two methods for extracting the bovine sperm RNA have residual organic solvent and protein. The OD value of RNA extracted by the Tiangen RNA extraction kit and the TRIzol method is 1.95-2.00, and almost pure RNA is obtained, so the method is determined to be the best scheme in the test.
RNA Integrity (RIN) is an important index for evaluating RNA quality, schuster et al prove that sperm RNA has higher quality of extracted RNA near RIN =3, RIN <2 indicates that a large amount of RNA is degraded, and RIN >4 indicates that somatic RNA pollution exists. In the above-mentioned experiments of the present invention, the TIANGEN kit + TRIzol (2) group showed the highest integrity, and it is assumed that β -mercaptoethanol affected the integrity of RNA. In addition, a large number of RNAs with RNA < 200nt are one of the RNA characteristics of mammalian sperm, and in this experiment, the electrophorogram of bovine sperm RNA was consistent with the above characteristics.
Claims (5)
1. A semen RNA extraction method comprises the following steps:
1) Adding the semen into an RNase-Free centrifuge tube, centrifuging to obtain sperm precipitate, resuspending the sperm precipitate in PBS, treating with RNAscope at 4 ℃ overnight, adding 2 times volume of precooled PBS, centrifuging at 4 ℃ by 5000 Xg for 5min, discarding the supernatant, and placing the sperm precipitate on ice for later use;
2) Adding RL lysate into the sperm precipitate, adding no beta-mercaptoethanol into the lysate, carrying out water bath at 60 ℃ for 3min until the solution is transparent, adding a TRIzol reagent with the same volume as the RL lysate into a centrifugal tube, carrying out vortex for 15s, standing for 3min, centrifuging for 15min at 4 ℃ of 15000 Xg, and taking supernatant;
3) Recovering the RNA.
2. The method of claim 1, wherein the step 3) of recovering RNA is by: adding equal volume of absolute ethyl alcohol into the supernatant obtained in the step 2), gently mixing uniformly, transferring into an RNase-Free adsorption column CR3 for adsorption, centrifuging to remove waste liquid, transferring the RNase-Free adsorption column CR3 into a collecting tube, adding deproteinizing liquid into the RNase-Free adsorption column CR3, centrifuging to remove waste liquid, adding rinsing liquid into the RNase-Free adsorption column CR3, standing at room temperature for 2min, centrifuging to remove waste liquid, repeatedly rinsing once, standing for air drying, transferring the RNase-Free adsorption column CR3 into a new RNase-Free centrifuge tube, adding RNase-Free ddH, and drying 2 And standing at room temperature for 2min, and centrifuging to obtain an RNA solution.
3. The method of claim 1, wherein the amount of lysis solution used in step 2) is 500 μ L.
4. The method of claim 2, wherein the step 3) of recovering RNA is by: adding equal volume of absolute ethyl alcohol into 500 mu L of supernate obtained in the step 2), gently mixing uniformly, transferring into an RNase-Free adsorption column CR3 for adsorption, centrifuging 30-60S to remove waste liquid at 13,400 Xg, transferring the RNase-Free adsorption column CR3 into a collecting tube, adding 700 mu L of deproteinizing liquid into the RNase-Free adsorption column CR3, centrifuging 30-60S to remove waste liquid at 13,400 Xg, adding rinsing liquid into the RNase-Free adsorption column CR3, standing at room temperature for 2min,13,400 Xg, centrifuging 30-60S to remove waste liquid, repeatedly rinsing once, standing and drying, transferring the RNase-Free adsorption column CR3 into a new RNase-Free centrifuge tube, adding RNase-Free ddH, and lightly mixing uniformly 2 O is left at room temperature for 2min, and centrifuged at 13,400 Xg for 2min to obtain an RNA solution.
5. The method according to any one of claims 1 to 4, wherein the semen is bovine semen.
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