Reverse transcription reaction solution and method for preparing cDNA
阅读说明：本技术 反转录反应液及制备cDNA的方法 (Reverse transcription reaction solution and method for preparing cDNA ) 是由 张双宇 于 2019-03-25 设计创作，主要内容包括：本发明提出了反转录反应液及制备cDNA的方法。本发明所提出的反转录反应液和制备cDNA的方法,反转录时可同时去除RNA样品中的残留基因组DNA,反转录反应操作简便、反应快速、反应效果好、可重复性高。(The invention provides a reverse transcription reaction liquid and a method for preparing cDNA. The reverse transcription reaction solution and the method for preparing cDNA provided by the invention can remove residual genome DNA in an RNA sample during reverse transcription, and the reverse transcription reaction has the advantages of simple operation, quick reaction, good reaction effect and high repeatability.)
1. A reverse transcription reaction solution, comprising:
0.1 to 0.3 mol/l of tris (hydroxymethyl) aminomethane;
40-60 mmol/l ammonium sulfate;
8-12 mmol/l magnesium chloride;
0.1 to 0.3 mol/l potassium chloride;
2-4 mmol/l dithiothreitol;
0.5 to 0.8 mmol/l calcium chloride;
100-300 mmol/l trehalose;
1-3 mmol/l of each deoxynucleoside triphosphate;
5-20 micromole/liter Oligo-dT15A primer;
30-60 micromole/liter of random primer;
25-75 units/microliter of murine leukemia virus reverse transcriptase;
1.5-3 units/microliter of an RNase inhibitor;
0.2-1.0 unit/microliter of DNase I;
1.5 to 3 percent of glycerol; and
the balance of water.
2. The reverse transcription reaction solution according to claim 1, wherein the pH of the reverse transcription reaction solution is 8.0 to 9.0, preferably the pH is 8.8.
3. The reverse transcription reaction solution according to claim 1, wherein the concentration of DNase I in the reverse transcription reaction solution is 0.5 unit/microliter.
4. The reverse transcription reaction solution according to claim 1, wherein Oligo-dT is present in the reverse transcription reaction solution15The concentration of the primer was 12.5. mu. mol/l;
preferably, the concentration of the random primer in the reverse transcription reaction solution is 50 micromole/liter;
preferably, the sequence of the random primer is NNNNNN.
5. The reverse transcription reaction solution according to claim 1, wherein the amount of potassium chloride is 0.2 mol/l, the amount of ammonium sulfate is 50 mmol/l, the amount of dithiothreitol is 3 mmol/l, the amount of calcium chloride is 0.65 mmol/l, and the amount of trehalose is 200 mmol/l; the amount of glycerol used was 2.7%.
6. Use of the reverse transcription reaction solution according to any one of claims 1 to 5 in a reverse transcription treatment.
7. A method of preparing cDNA, comprising:
subjecting an RNA sample to reverse transcription treatment using the reverse transcription reaction solution according to any one of claims 1 to 5 to obtain the cDNA.
8. The method of claim 7, wherein the reverse transcription process is performed at 42 ℃ for 15 minutes.
9. The method of reverse transcription according to claim 8, further comprising:
(1) mixing the RNA sample, water and the reverse transcription reaction solution so as to obtain a reaction mixture, wherein the reverse transcription reaction solution is used in an amount of 4 microliters based on 10 nanograms to 2 micrograms of the RNA sample, and the water is used for adjusting the volume of the reaction mixture to 20 microliters; and
(2) subjecting the reaction mixture obtained in step (1) to the reverse transcription treatment so as to perform rapid reverse transcription and obtain cDNA.
10. The method of reverse transcription according to claim 9, wherein the water is deionized water.
11. The method of reverse transcription according to claim 10, wherein the mixing is performed at 4 degrees celsius.
Technical Field
The invention relates to the field of biotechnology, in particular to a reverse transcription reaction liquid and a method for preparing cDNA.
Background
Analysis and detection of gene expression and regulation is an important topic in the fields of modern molecular biology and medical research. With the development of gene expression regulation analysis detection technology in recent years, there are more and more related detection technologies, such as: in situ hybridization, Northern hybridization, RNase protection assay, gene chip analysis, real-time quantitative PCR analysis, reverse transcription PCR analysis (RT-PCR), and the like. Among them, RT-PCR analysis is considered to be one of the main means for detecting gene expression because of its simple operation and sensitive mRNA level detection. RT-PCR is a detection technique that combines Reverse Transcription (RT) reaction and PCR (polymerase Chain reaction) reaction. The RT (reverse transcription) reaction is the key of RT-PCR technology, and is a process of synthesizing DNA (complementary deoxyribonucleic acid) complementary to mRNA of a template by using mRNA as the template and under the action of reverse transcriptase.
cDNA synthesis and cloning techniques have become important tools for molecular biology research today. The first strand of cDNA is synthesized without the action of RNA-dependent DNA polymerases (reverse transcriptases). Reverse transcriptases which are very commonly used and have been commercialized at present are both Avian Myeloblastosis Virus (AMV) reverse transcriptase and murine leukemia virus (MMLV) reverse transcriptase. In general, the RT reaction process consists of 3 steps: firstly, opening a secondary structure of an RNA template through high temperature; secondly, catalyzing reverse transcription reaction by reverse transcriptase; finally, the reverse transcriptase is inactivated through high temperature so as to ensure the smooth proceeding of the subsequent PCR process. Therefore, the RT reaction step in the conventional method is complicated.
In addition, DNA in a sample is extracted at the same time in the process of extracting RNA, and residual DNA interferes with the detection effect of cDNA which is a reverse transcription product of the RNA in the subsequent PCR detection reaction. For such genomic DNA, it can generally be removed by two methods: one is to add a DNA digestion step in the RNA extraction process; the other is to add a DNA digestion step during reverse transcription of RNA. However, since RNA itself is not stable and is easily degraded, any of the above methods for removing genomic DNA will increase the operation steps in the RNA extraction or reverse transcription process, which increases the risk of RNA degradation, and reduces the reverse transcription effect of RNA while complicating the operation process. Therefore, if the genomic DNA removal reaction and the RNA reverse transcription reaction can be integrated, so that the genomic DNA removal process can be completed while the RNA reverse transcription reaction is carried out, the operation steps of the reverse transcription can be simplified, and the reverse transcription effect of the RNA can be improved.
Therefore, how to further simplify the reverse transcription step of RNA and improve the efficiency of cDNA synthesis still remains to be developed.
Disclosure of Invention
The present invention is directed to solving, at least to some extent, one of the technical problems in the related art.
The present invention has been completed based on the following findings of the inventors:
the conventional operation of reverse transcription requires 3 different reaction temperatures (42 ℃, 65 ℃ and 95 ℃), and the preparation process of the reaction system is complicated and error-prone, and in addition, the whole operation process requires approximately 2 hours of operation and reaction time, and if a removal step of genomic DNA is required, the operation steps and operation time are increased, so that the conventional operation process is time-consuming and labor-consuming. Therefore, the conventional reverse transcription reaction not only brings inconvenience to the experimental operator, but also cannot meet the requirements of high speed and high flux of gene analysis.
At present, some commercial kits are lack of products which can simultaneously remove genome DNA and reverse transcription, and have rapid reaction and simple and convenient operation, but the effects of removing the genome and the reverse transcription are general, the repeatability is not good, and the popularization is difficult.
Based on the above problems, the present inventors have proposed a reverse transcription reaction solution capable of removing genomic DNA simultaneously, and the reverse transcription reaction solution can be used to perform reverse transcription on RNA samples, so as to remove genomic DNA and perform reverse transcription simultaneously, and the time is only 15 minutes, the removal of genomic DNA is complete, the reverse transcription reaction effect is good, and the repeatability is good.
In a first aspect of the present invention, a reverse transcription reaction solution is provided. According to an embodiment of the present invention, the reverse transcription reaction solution comprises: 0.1 to 0.3 mol/l of tris (hydroxymethyl) aminomethane; 40-60 mmol/l ammonium sulfate; 8-12 mmol/l magnesium chloride; 0.1 to 0.3 mol/l potassium chloride; 2-4 mmol/l dithiothreitol; 0.5-0.8 mol/l calcium chloride; 100-300 mmol/l trehalose; deoxynucleoside triphosphates (including dATP, dTTP, dGTP and dCTP) are 1-3 mmol/L respectively; 5-20 micromole/liter Oligo-dT15A primer; 30-60 micromole/liter of random primer; 25-75 units/Microliter of Murine Leukemia Virus (MMLV) reverse transcriptase; 1.5-3 units/microliter of an RNase inhibitor; 0.2-1.0 unit/microliter of DNase I (DNase I); 1.5-3% (volume ratio) of glycerol; and the balance water. According to the embodiment of the invention, the reverse transcription reaction liquid provided by the invention is used for reverse transcription, residual genome DNA in an RNA sample can be removed simultaneously during reverse transcription, and the reverse transcription reaction is simple and convenient to operate, quick in reaction, good in reaction effect and high in repeatability.
According to an embodiment of the present invention, the reverse transcription reaction solution may further have one of the following additional features:
according to an embodiment of the present invention, the Oligo-dT15The sequence of the primer was TTTTTTTTTTTTTTT.
According to an embodiment of the invention, the sequence of the random primer is NNNNNN. Wherein N represents any one of A, T, C, G bases, and the random primer is composed of a sequence comprising 6 arbitrary consecutive repeated bases.
According to the embodiment of the invention, the pH of the reverse transcription reaction liquid is 8.0-9.0. When the pH value is 8.0-9.0, the MMLV reverse transcriptase utilizes the RNA template to synthesize cDNA, so that the activity and the efficiency are high. According to an embodiment of the present invention, it is preferable that the pH of the reverse transcription reaction solution is 8.8. The inventor finds that under the condition of pH being 8.8, the DNAseI can play the maximum function in the reverse transcription process, the activity of synthesizing cDNA by MMLV reverse transcriptase by utilizing an RNA template is played to the maximum extent, and the function of removing residual genome and the function of reverse transcription are both realized. The reverse transcription reaction liquid of the embodiment of the invention is utilized for reverse transcription, the reverse transcription reaction speed is further improved, and the reaction effect and the repeatability are further improved.
According to the embodiment of the invention, the concentration of DNase I in the reverse transcription reaction solution is 0.2-1.0 unit/microliter. In the development process, the inventor finds that if the genome removal function and the reverse transcription function are integrated in the reverse transcription process, the influence of DNase I on the reverse transcription is considered, and the influence is only expressed in two aspects, on one hand, DNase I can cut reverse transcription primers in a reaction system to influence the reverse transcription efficiency, and on the other hand, DNase I can degrade newly reverse transcribed cDNA to influence the reverse transcription yield. The amount of DNase I used in the reaction mixture of the present invention was selected based on the knowledge of various problems of the present inventors. The inventor finds that when the concentration of DNase I is 0.2-1.0 unit/microliter, in the reverse transcription process, DNase I can remove residual genome DNA, does not cause loss to the effective concentration of a primer (single-stranded DNA) in a reverse transcription reaction liquid and newly synthesized cDNA, can realize that the genome removing process and the reverse transcription process are simultaneously carried out in one tube, simplifies the reverse transcription process and improves the reverse transcription effect.
According to the embodiment of the present invention, it is preferable that the concentration of DNase I in the reverse transcription reaction solution is 0.5 unit/microliter, so that residual genomic DNA can be removed to the maximum extent, and the activity of MMLV reverse transcriptase in synthesizing cDNA using RNA template can be exerted to the maximum extent. The reverse transcription reaction liquid of the embodiment of the invention is utilized for reverse transcription, the reverse transcription reaction speed, the genome DNA removal effect and convenience are further improved, and the reaction effect and repeatability are further improved.
According to the embodiment of the invention, the concentration of the random primer of the reverse transcription reaction solution is 30-60 micromoles/liter; Oligo-dT15The concentration of the primer is 5-20 micromoles/liter. In the research and development process, the inventor finds that the effective concentrations of the two reverse transcription primers have obvious influence on the reverse transcription efficiency, and the reverse transcription reaction liquid involved in the invention contains DNase I which degrades the two reverse transcription primers, so that the addition amount of the two reverse transcription primers is greatly increased compared with the conventional reverse transcription reaction liquid, the degradation reaction of DNase I on random primers is effectively inhibited, and the effective concentration of the reverse transcription primers in the reverse transcription reaction is ensured. The inventor finds that the concentration of the random primer is 30-60 micromoles/liter; Oligo-dT15Under the condition that the concentration of the primer is 5-20 micromoles/liter, the random primer and Oligo-dT can not be subjected to primer pair even if DNase I exists in a reverse transcription reaction solution15The effective concentration of the primer is lost, and the feasibility of simultaneous genome removal and reverse transcription in one tube is ensured in another aspect.
According to embodiments of the present invention, it is preferred that the concentration of random primers is 50. mu.M, Oligo-dT15The concentration of the primer is 12.5 micromole/liter, the activity of synthesizing cDNA by MMLV reverse transcriptase by utilizing an RNA template is exerted to the maximum extent, so that the reverse transcription reaction liquid is utilized for reverse transcription, and the reverse transcription reaction speed, the reaction effect and the repeatability are further improved.
According to an embodiment of the invention, the amount of potassium chloride is 0.2 mmol/l, the amount of ammonium sulphate is 50 mmol/l, the amount of dithiothreitol is 3 mmol/l, the amount of calcium chloride is 0.65 mmol/l, the amount of trehalose is 200 mmol/l; the amount of glycerol used was 2.7%. The invention finds that if the genome removing step and the reverse transcription step are integrated in the reverse transcription process, the cation species and proportion in the reaction liquid and the addition proportion of the protective agent can simultaneously satisfy the two reactions. According to the embodiment of the invention, under the condition of using the potassium chloride, the ammonium sulfate, the dithiothreitol, the calcium chloride, the trehalose and the glycerol, the genome removing effect, the reverse transcription reaction speed, the reaction effect and the repeatability of the reverse transcription reaction liquid are further and remarkably improved.
In a second aspect of the present invention, the present invention provides use of the aforementioned reverse transcription reaction solution in a reverse transcription treatment. According to the embodiment of the invention, the reverse transcription reaction liquid provided by the invention is utilized for reverse transcription, the reverse transcription reaction is simple and convenient to operate, the residual genome DNA in an RNA sample can be effectively removed in the reverse transcription treatment process, the reaction is rapid, the reaction effect is good, and the repeatability is high.
In a third aspect of the invention, a method of preparing a cDNA is provided. According to an embodiment of the invention, the method comprises: the RNA sample was subjected to reverse transcription treatment using the reverse transcription reaction solution described above to obtain the cDNA. According to the embodiment of the invention, the method for preparing cDNA provided by the invention has the same effect and advantages as the reverse transcription reaction solution provided by the invention, namely the method for preparing cDNA provided by the invention can realize the effective removal of residual genome DNA in an RNA sample, and simultaneously can quickly realize the acquisition of cDNA, and has the advantages of quick reaction, good effect and high repeatability.
According to an embodiment of the present invention, the above method for preparing cDNA may further have one of the following additional technical features:
according to the embodiment of the present invention, the reverse transcription process (the process of removing residual genomic DNA from the RNA-containing sample) is performed at 42 ℃ for 15 minutes. The time required by the conventional reverse transcription treatment is 60-65 minutes, the method for preparing cDNA provided by the invention is 15 minutes, and the reaction is rapid.
According to an embodiment of the invention, the method comprises: (1) mixing the RNA sample, water and the reverse transcription reaction solution so as to obtain a reaction mixture, wherein the reverse transcription reaction solution is used in an amount of 4 microliters based on 10 nanograms to 2 micrograms of the RNA sample, and the water is used for adjusting the volume of the reaction mixture to 20 microliters; and (2) subjecting the reaction mixture obtained in step (1) to the reverse transcription treatment (a residual genomic DNA removal process from the RNA-containing sample) to obtain the cDNA. According to the embodiment of the invention, the method for preparing cDNA provided by the invention can simultaneously realize the effective removal of residual genome DNA in an RNA sample in the process of RNA reverse transcription, and has the characteristics of quick reaction, good reaction effect and high repeatability.
According to an embodiment of the invention, the water is deionized water. The deionized water can prevent the RNase from degrading RNA, prevent ions in water from interfering a reaction system and avoid the generation of non-specific products. Therefore, the use of deionized water during the reverse transcription process further improves the stability and reproducibility of the method for preparing cDNA according to the present invention.
According to an embodiment of the invention, the mixing is performed at 4 degrees celsius. The mixing treatment is carried out at 4 ℃, so that the stability of the RNA sample and the stability of the reverse transcriptase in the reaction system are ensured. Therefore, the mixing process is performed at 4 degrees celsius to ensure further improvement in stability and reliability of the method for preparing cDNA proposed by the present invention.
Drawings
FIG. 1 is a view of fluorescent quantitative PCR of cDNA prepared by the method for preparing cDNA according to the present invention according to example 10 of the present invention;
FIG. 2 is a view showing fluorescent quantitative PCR detection of cDNA prepared by a conventional reverse transcription method according to example 10 of the present invention;
FIG. 3 is a graph showing the results of electrophoresis in which cDNA prepared by the method for preparing cDNA according to the present invention and conventional reverse transcription method according to example 12 of the present invention were separately subjected to PCR detection, and then the PCR products were subjected to agarose gel electrophoresis.
Detailed Description
The following describes embodiments of the present invention in detail. The following examples are illustrative only and are not to be construed as limiting the invention.
The present invention has been completed based on the following findings of the inventors:
the conventional reverse transcription operation process needs 3 different reaction temperatures, the preparation process of a reaction system is complex, the whole operation process needs about 2 hours of operation and reaction time, and time and labor are consumed; if the step of removing the residual genome is added, the method becomes more complicated and time-consuming, and some commercial kits have general reaction effect and poor repeatability at present.
Based on the problems, the inventor provides a reverse transcription reaction solution which is simple and convenient to operate and rapid in reaction, residual genome in an RNA sample is removed simultaneously in the process of carrying out reverse transcription treatment on the RNA sample by using the reverse transcription reaction solution, the time required by the whole reverse transcription process is only 15 minutes, the reverse transcription reaction effect is good, and the repeatability is good.
Reverse transcription reaction liquid
In a first aspect of the present invention, a reverse transcription reaction solution is provided. According to an embodiment of the present invention, the reverse transcription reaction solution comprises: 0.1 to 0.3 mol/l of tris (hydroxymethyl) aminomethane; 40-60 mmol/l ammonium sulfate; 8-12 mmol/l magnesium chloride; 0.1 to 0.3 mol/l potassium chloride; 2-4 mmol/l dithiothreitol; 0.5-0.8 mol/l calcium chloride; 100-300 mmol/l trehalose; 1-3 mmol/l of each deoxynucleoside triphosphate; 5-20 micromole/liter Oligo-dT15A primer; 30-60 micromole/liter of random primer; 25-75 units/Microliter of Murine Leukemia Virus (MMLV) reverse transcriptase; 1.5-3 units/microliter of an RNase inhibitor; 0.2-1.0 unit/microliter of DNase I (DNase I); 1.5-3% (volume ratio) of glycerol; and the balance water.
The inventor finds that the addition of 100-300 mmol/L trehalose and 1.5% -3% glycerol in the reverse transcription reaction solution can well protect the effects of reverse transcriptase, DNase I, RNase inhibitor and ultrapure dNTPs, so that the reverse transcription reaction solution still has high activity under the conditions of freezing and thawing for 30 times and freezing and thawing at-20 ℃ for 6 months. Meanwhile, the inventor surprisingly finds that the reverse transcription reaction liquid is added with 40-60 mmol/L ammonium sulfate and 0.1-0.3 mol/L potassium chloride, and the proportion of ammonium ions and potassium ions can ensure the specific and rapid combination of a reverse transcription primer and template RNA, so that the reverse transcription reaction is carried out efficiently, DNase I can normally exert the effect, and residual genome is removed without influencing the reverse transcription. And 2-4 mmol/L Dithiothreitol (DTT) is added into the reverse transcription reaction liquid, so that the reaction rate of the reverse transcriptase is increased. Finally, the inventors have surprisingly found that 0.2 to 1.0 unit/microliter of DNase I (DNase I) and a high concentration of reverse transcription primer (5 to 20. mu.M of Oligo-dT) are added to the reaction solution for reverse transcription15A primer; 30-60 micromole/liter random primer), the purpose of degrading residual genome DNA in an RNA sample can be realized, the effective concentration of a reverse transcription primer in a reverse transcription reaction solution cannot be influenced, and simultaneously, cDNA synthesized by new reverse transcription cannot be damaged. The genome removing reaction and the reverse transcription reaction can be simultaneously carried out in one tube, and the effect is stable and the reverse transcription effect is good.
According to the embodiment of the invention, the reverse transcription reaction liquid provided by the invention is utilized for reverse transcription, the reverse transcription reaction is simple and convenient to operate, the reaction is rapid, the reaction effect is good, and the repeatability is high.
According to the embodiment of the invention, the pH of the reverse transcription reaction liquid is 8.0-9.0. When the pH value is 8.0-9.0, the MMLV reverse transcriptase utilizes the RNA template to synthesize cDNA, so that the activity and the efficiency are high. According to the embodiment of the present invention, the pH of the reverse transcription reaction solution is preferably 8.8, and the reverse transcription reaction solution has both the residual genome removal function and the reverse transcription function, so that the pH environment of the reaction solution needs to be taken into consideration, and experimental results show that the activity of the MMLV reverse transcriptase for synthesizing cDNA by using the RNA template can be maximally exerted when the pH is 8.8. The reverse transcription reaction liquid of the embodiment of the invention is utilized for reverse transcription, the reverse transcription reaction speed is further improved, and the reaction effect and the repeatability are further improved.
According to the embodiment of the invention, the concentration of DNase I in the reverse transcription reaction solution is 0.2-1.0 unit/microliter. The integration of the genome removal step with the reverse transcription step requires consideration of the effect of DNase i on reverse transcription, which is mainly reflected in two aspects: on one hand, DNase I can cut reverse transcription primers in a reaction system to influence the reverse transcription efficiency; on the other hand, DNase I degrades newly reverse transcribed cDNA, thereby affecting reverse transcription yield. Therefore, the amount of DNase I used in the reaction mixture of the present invention is selected in consideration of various factors. When the concentration of DNase I is 0.2-1.0 unit/microliter, in the reverse transcription process, DNase I can remove residual genome DNA, and does not cause loss to the effective concentration of a primer (single-stranded DNA) in a reverse transcription reaction solution and newly synthesized cDNA, so that the genome removing process and the reverse transcription process can be simultaneously carried out in one tube, the reverse transcription process is simple and convenient, and the reverse transcription effect is improved.
According to the embodiment of the present invention, it is preferable that the concentration of DNase I in the reverse transcription reaction solution is 0.5 unit/microliter, so that residual genomic DNA can be removed to the maximum extent, and the activity of MMLV reverse transcriptase in synthesizing cDNA using RNA template can be exerted to the maximum extent. The reverse transcription reaction liquid of the embodiment of the invention is utilized for reverse transcription, the reverse transcription reaction speed, the genome DNA removal effect and convenience are further improved, and the reaction effect and repeatability are further improved.
According to the embodiment of the invention, the concentration of the random primer of the reverse transcription reaction solution is 30-60 micromoles/liter; the concentration of Oligo-dT15 primer is 5-20 μmol/L. The effective concentration of the two reverse transcription primers is critical to the reverse transcription efficiency. As the reverse transcription reaction liquid related in the invention contains DNase I which can degrade the two reverse transcription primers, the addition amount of the two reverse transcription primers is greatly increased compared with the conventional reverse transcription reaction liquid for the reaction liquid, so as to inhibit the degradation reaction of the DNase I on random primers and ensure the effective concentration of the reverse transcription primers in the reverse transcription reaction. The concentration of the random primer is 30-60 micromoles/liter; Oligo-dT15Under the condition that the concentration of the primer is 5-20 micromoles/liter, the effective concentrations of the random primer and the Oligo-dT15 primer cannot be lost even if DNase I exists in a reverse transcription reaction liquid, and the feasibility of simultaneous implementation of a genome removal process and a reverse transcription process in one tube is ensured from the other aspect.
According to embodiments of the present invention, it is preferred that the concentration of random primers is 50. mu.M, Oligo-dT15The concentration of the primer is 12.5 micromole/liter, the activity of synthesizing cDNA by MMLV reverse transcriptase by utilizing an RNA template is exerted to the maximum extent, so that the reverse transcription reaction liquid is utilized for reverse transcription, and the reverse transcription reaction speed, the reaction effect and the repeatability are further improved.
According to an embodiment of the invention, the amount of potassium chloride is 0.2 mmol/l, the amount of ammonium sulphate is 50 mmol/l, the amount of dithiothreitol is 3 mmol/l, the amount of calcium chloride is 0.65 mmol/l, the amount of trehalose is 200 mmol/l; the amount of glycerol used was 2.7%. For the reverse transcription reaction solution integrating the genome removal step into the reverse transcription step, the species and the proportion of cations in the reaction solution and the addition ratio of the protective agent are also needed to take care of the two reactions, so that the genome removal effect, the reverse transcription reaction speed, the reaction effect and the repeatability of the reverse transcription reaction solution are further remarkably improved by utilizing the reverse transcription reaction solution of the embodiment of the invention to carry out reverse transcription under the use amount of the potassium chloride, the ammonium sulfate, the dithiothreitol, the calcium chloride, the trehalose and the glycerol.
Method for preparing cDNA
In another aspect of the invention, the invention features a method of preparing a cDNA. According to an embodiment of the invention, comprising: the RNA sample was subjected to reverse transcription treatment using the reverse transcription reaction solution described above to obtain the cDNA.
According to an embodiment of the present invention, the reverse transcription process is performed at 42 ℃ for 15 minutes.
The method for preparing cDNA provided by the invention adopts four-step reaction (genome removing step, RNA advanced structure opening step, reverse transcription reaction step and enzyme heat inactivation step) as one-step reaction, and shortens the reaction time of nearly two hours to 15 minutes. This method not only eliminates the initial steps of residual genomic DNA digestion, pre-denaturation of the RNA template and final reverse transcriptase denaturation, but also saves at least 70% of the reverse transcription time, as compared to conventional methods. Greatly saving the operation time of experiment operators. According to the embodiment of the invention, the method for preparing cDNA provided by the invention has the characteristics of rapid reverse transcription reaction, good effect and high repeatability.
According to an embodiment of the present invention, the reverse transcription process further comprises: (1) mixing the RNA sample, water and the reverse transcription reaction solution so as to obtain a reaction mixture, wherein the reverse transcription reaction solution is used in an amount of 4 microliters based on 10 nanograms to 2 micrograms of the RNA sample, and the water is used for adjusting the volume of the reaction mixture to 20 microliters; and (2) subjecting the reaction mixture obtained in step (1) to the reverse transcription treatment to obtain the cDNA. In the method for preparing cDNA provided by the invention, 7 simplified components are 1 component, namely 5 times reverse transcription reaction liquid is used for replacing the following 7 components in the conventional operation method: the reverse transcription reaction comprises a reverse transcription buffer solution, a reverse transcription primer, a reverse transcriptase, ultrapure dNTPs, an RNase inhibitor, DNae I and DNae I reaction buffer solution, wherein an experimenter does not need to separately add the 7 components, water and RNA step by step in the reverse transcription operation process, but only needs to add water, 5 times of reverse transcription reaction solution and a proper amount of RNA. The method not only greatly saves the operation time of experimenters, but also avoids the possibility of cross contamination among samples caused by multiple step-by-step operations, so that the reverse transcription result is more accurate and reliable, and the method is greatly convenient for scientific research personnel in large-scale operation. Therefore, the method for preparing cDNA provided by the invention has the characteristics of simple and convenient operation and quick reaction.
According to an embodiment of the present invention, the water is deionized water. The deionized water can avoid the degradation of RNase in water to RNA, the interference of ions in water to a reaction system and the generation of non-specific products. Therefore, the use of deionized water during the reverse transcription process further improves the stability and reproducibility of the method for preparing cDNA according to the present invention.
According to an embodiment of the invention, the mixing is performed at 4 degrees celsius. The mixing treatment is carried out at 4 ℃, so that the stability of the RNA sample and the stability of the reverse transcriptase in the reaction system are ensured. Therefore, the mixing process is performed at 4 degrees celsius to ensure further improvement in stability and reliability of the method for preparing cDNA proposed by the present invention.
The scheme of the invention will be explained with reference to the examples. It will be appreciated by those skilled in the art that the following examples are illustrative of the invention only and should not be taken as limiting the scope of the invention. The examples, where specific techniques or conditions are not indicated, are to be construed according to the techniques or conditions described in the literature in the art or according to the product specifications. The reagents or instruments used are not indicated by manufacturers, and all are conventional products which can be obtained by purchasing.
In the examples of the present invention, the TIANCcript II cDNA first strand synthesis kit, FastKing cDNA first strand synthesis kit, SuperReal PreMix (SYBR Green) fluorescent quantitative detection kit and TIANseq HiFi Amplification Mix used were provided by Tiangen Biochemical technology (Beijing) Ltd. The primer probe used was synthesized by Invitrogen, USA.