CN111349693A - Color reverse transcription premixed liquid - Google Patents
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- CN111349693A CN111349693A CN201811561205.1A CN201811561205A CN111349693A CN 111349693 A CN111349693 A CN 111349693A CN 201811561205 A CN201811561205 A CN 201811561205A CN 111349693 A CN111349693 A CN 111349693A
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- 238000010839 reverse transcription Methods 0.000 title claims abstract description 76
- 239000007788 liquid Substances 0.000 title abstract description 10
- 239000000243 solution Substances 0.000 claims description 22
- 102100034343 Integrase Human genes 0.000 claims description 18
- 108010092799 RNA-directed DNA polymerase Proteins 0.000 claims description 18
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 claims description 9
- TWRXJAOTZQYOKJ-UHFFFAOYSA-L Magnesium chloride Chemical compound [Mg+2].[Cl-].[Cl-] TWRXJAOTZQYOKJ-UHFFFAOYSA-L 0.000 claims description 6
- WCUXLLCKKVVCTQ-UHFFFAOYSA-M Potassium chloride Chemical compound [Cl-].[K+] WCUXLLCKKVVCTQ-UHFFFAOYSA-M 0.000 claims description 6
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 claims description 4
- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 claims description 4
- SEQKRHFRPICQDD-UHFFFAOYSA-N N-tris(hydroxymethyl)methylglycine Chemical compound OCC(CO)(CO)[NH2+]CC([O-])=O SEQKRHFRPICQDD-UHFFFAOYSA-N 0.000 claims description 4
- 239000000872 buffer Substances 0.000 claims description 4
- 159000000003 magnesium salts Chemical class 0.000 claims description 4
- 239000003161 ribonuclease inhibitor Substances 0.000 claims description 4
- 241000713869 Moloney murine leukemia virus Species 0.000 claims description 3
- 229910001629 magnesium chloride Inorganic materials 0.000 claims description 3
- XAEFZNCEHLXOMS-UHFFFAOYSA-M potassium benzoate Chemical compound [K+].[O-]C(=O)C1=CC=CC=C1 XAEFZNCEHLXOMS-UHFFFAOYSA-M 0.000 claims description 3
- 239000001103 potassium chloride Substances 0.000 claims description 3
- 235000011164 potassium chloride Nutrition 0.000 claims description 3
- JKMHFZQWWAIEOD-UHFFFAOYSA-N 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid Chemical compound OCC[NH+]1CCN(CCS([O-])(=O)=O)CC1 JKMHFZQWWAIEOD-UHFFFAOYSA-N 0.000 claims description 2
- 239000007995 HEPES buffer Substances 0.000 claims description 2
- BELBBZDIHDAJOR-UHFFFAOYSA-N Phenolsulfonephthalein Chemical compound C1=CC(O)=CC=C1C1(C=2C=CC(O)=CC=2)C2=CC=CC=C2S(=O)(=O)O1 BELBBZDIHDAJOR-UHFFFAOYSA-N 0.000 claims description 2
- UZMAPBJVXOGOFT-UHFFFAOYSA-N Syringetin Natural products COC1=C(O)C(OC)=CC(C2=C(C(=O)C3=C(O)C=C(O)C=C3O2)O)=C1 UZMAPBJVXOGOFT-UHFFFAOYSA-N 0.000 claims description 2
- 239000007997 Tricine buffer Substances 0.000 claims description 2
- 208000034953 Twin anemia-polycythemia sequence Diseases 0.000 claims description 2
- OWMVSZAMULFTJU-UHFFFAOYSA-N bis-tris Chemical compound OCCN(CCO)C(CO)(CO)CO OWMVSZAMULFTJU-UHFFFAOYSA-N 0.000 claims description 2
- OBRMNDMBJQTZHV-UHFFFAOYSA-N cresol red Chemical compound C1=C(O)C(C)=CC(C2(C3=CC=CC=C3S(=O)(=O)O2)C=2C=C(C)C(O)=CC=2)=C1 OBRMNDMBJQTZHV-UHFFFAOYSA-N 0.000 claims description 2
- 239000013078 crystal Substances 0.000 claims description 2
- KCFYHBSOLOXZIF-UHFFFAOYSA-N dihydrochrysin Natural products COC1=C(O)C(OC)=CC(C2OC3=CC(O)=CC(O)=C3C(=O)C2)=C1 KCFYHBSOLOXZIF-UHFFFAOYSA-N 0.000 claims description 2
- 229910052943 magnesium sulfate Inorganic materials 0.000 claims description 2
- 235000019341 magnesium sulphate Nutrition 0.000 claims description 2
- PGSADBUBUOPOJS-UHFFFAOYSA-N neutral red Chemical compound Cl.C1=C(C)C(N)=CC2=NC3=CC(N(C)C)=CC=C3N=C21 PGSADBUBUOPOJS-UHFFFAOYSA-N 0.000 claims description 2
- 229960003531 phenolsulfonphthalein Drugs 0.000 claims description 2
- OTYBMLCTZGSZBG-UHFFFAOYSA-L potassium sulfate Chemical compound [K+].[K+].[O-]S([O-])(=O)=O OTYBMLCTZGSZBG-UHFFFAOYSA-L 0.000 claims description 2
- 229910052939 potassium sulfate Inorganic materials 0.000 claims description 2
- 235000011151 potassium sulphates Nutrition 0.000 claims description 2
- 239000004094 surface-active agent Substances 0.000 claims description 2
- CTQNGGLPUBDAKN-UHFFFAOYSA-N O-Xylene Chemical compound CC1=CC=CC=C1C CTQNGGLPUBDAKN-UHFFFAOYSA-N 0.000 claims 1
- 239000008096 xylene Substances 0.000 claims 1
- 238000006243 chemical reaction Methods 0.000 abstract description 55
- 238000011529 RT qPCR Methods 0.000 abstract description 28
- 238000002360 preparation method Methods 0.000 abstract description 19
- 238000001514 detection method Methods 0.000 abstract description 10
- 238000003757 reverse transcription PCR Methods 0.000 abstract description 5
- 239000011259 mixed solution Substances 0.000 abstract 1
- 239000002299 complementary DNA Substances 0.000 description 24
- 239000000975 dye Substances 0.000 description 22
- 238000000034 method Methods 0.000 description 15
- 239000000523 sample Substances 0.000 description 13
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 10
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 8
- 239000000203 mixture Substances 0.000 description 8
- 238000002156 mixing Methods 0.000 description 5
- 108020004999 messenger RNA Proteins 0.000 description 4
- 239000011535 reaction buffer Substances 0.000 description 4
- 241000713838 Avian myeloblastosis virus Species 0.000 description 3
- 238000002474 experimental method Methods 0.000 description 3
- IKHKJYWPWWBSFZ-UHFFFAOYSA-N 4-[[4-(diethylamino)phenyl]-(4-diethylazaniumylidenecyclohexa-2,5-dien-1-ylidene)methyl]benzene-1,3-disulfonate;hydron Chemical compound C1=CC(N(CC)CC)=CC=C1C(C=1C(=CC(=CC=1)S([O-])(=O)=O)S(O)(=O)=O)=C1C=CC(=[N+](CC)CC)C=C1 IKHKJYWPWWBSFZ-UHFFFAOYSA-N 0.000 description 2
- 239000012807 PCR reagent Substances 0.000 description 2
- SUYVUBYJARFZHO-RRKCRQDMSA-N dATP Chemical compound C1=NC=2C(N)=NC=NC=2N1[C@H]1C[C@H](O)[C@@H](COP(O)(=O)OP(O)(=O)OP(O)(O)=O)O1 SUYVUBYJARFZHO-RRKCRQDMSA-N 0.000 description 2
- SUYVUBYJARFZHO-UHFFFAOYSA-N dATP Natural products C1=NC=2C(N)=NC=NC=2N1C1CC(O)C(COP(O)(=O)OP(O)(=O)OP(O)(O)=O)O1 SUYVUBYJARFZHO-UHFFFAOYSA-N 0.000 description 2
- RGWHQCVHVJXOKC-SHYZEUOFSA-J dCTP(4-) Chemical compound O=C1N=C(N)C=CN1[C@@H]1O[C@H](COP([O-])(=O)OP([O-])(=O)OP([O-])([O-])=O)[C@@H](O)C1 RGWHQCVHVJXOKC-SHYZEUOFSA-J 0.000 description 2
- HAAZLUGHYHWQIW-KVQBGUIXSA-N dGTP Chemical compound C1=NC=2C(=O)NC(N)=NC=2N1[C@H]1C[C@H](O)[C@@H](COP(O)(=O)OP(O)(=O)OP(O)(O)=O)O1 HAAZLUGHYHWQIW-KVQBGUIXSA-N 0.000 description 2
- NHVNXKFIZYSCEB-XLPZGREQSA-N dTTP Chemical compound O=C1NC(=O)C(C)=CN1[C@@H]1O[C@H](COP(O)(=O)OP(O)(=O)OP(O)(O)=O)[C@@H](O)C1 NHVNXKFIZYSCEB-XLPZGREQSA-N 0.000 description 2
- 230000014509 gene expression Effects 0.000 description 2
- IZMJMCDDWKSTTK-UHFFFAOYSA-N quinoline yellow Chemical compound C1=CC=CC2=NC(C3C(C4=CC=CC=C4C3=O)=O)=CC=C21 IZMJMCDDWKSTTK-UHFFFAOYSA-N 0.000 description 2
- 229940051201 quinoline yellow Drugs 0.000 description 2
- 235000012752 quinoline yellow Nutrition 0.000 description 2
- 239000004172 quinoline yellow Substances 0.000 description 2
- 108091032955 Bacterial small RNA Proteins 0.000 description 1
- -1 DTT Substances 0.000 description 1
- 241000714177 Murine leukemia virus Species 0.000 description 1
- 108091034117 Oligonucleotide Proteins 0.000 description 1
- 229920001213 Polysorbate 20 Polymers 0.000 description 1
- 239000013614 RNA sample Substances 0.000 description 1
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- 239000012154 double-distilled water Substances 0.000 description 1
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- 239000000256 polyoxyethylene sorbitan monolaurate Substances 0.000 description 1
- 235000010486 polyoxyethylene sorbitan monolaurate Nutrition 0.000 description 1
- 229920000136 polysorbate Polymers 0.000 description 1
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- 159000000001 potassium salts Chemical class 0.000 description 1
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- 238000003753 real-time PCR Methods 0.000 description 1
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- GPRLSGONYQIRFK-MNYXATJNSA-N triton Chemical compound [3H+] GPRLSGONYQIRFK-MNYXATJNSA-N 0.000 description 1
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Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6844—Nucleic acid amplification reactions
- C12Q1/686—Polymerase chain reaction [PCR]
Abstract
The invention provides a color reverse transcription premix liquid. The pre-mixed solution contains all components of the reverse transcription reaction except the template and also contains dye. The color reverse transcription premixed liquid can greatly simplify the preparation steps of a reverse transcription reaction system, track the preparation process of the reverse transcription and real-time quantitative PCR reaction system in real time and judge which components are added into the reaction system, thereby greatly reducing the possibility of experimental error and improving the experimental detection efficiency.
Description
Technical Field
The invention relates to the field of molecular biology, in particular to a reverse transcription premix.
Background
Reverse transcription PCR (RT-PCR) is a current general method for detecting gene expression level in cells/tissues, so that the sensitivity of RNA detection is improved by several orders of magnitude, and the analysis of some extremely trace RNA samples becomes possible. In this technique, mRNA is first synthesized into cDNA by reverse transcriptase; then, PCR is carried out by using the cDNA as a template, and the detection of mRNA is realized by analyzing the PCR result. In the intensive study of gene expression levels in cells/tissues, a reverse transcription-real-time quantitative PCR (real-time PCR) method is often employed.
Generally, a Reverse Transcription (RT) reaction system is composed of the following components:
1. template (template): the template for reverse transcription is typically total RNA or mRNA. At present, with the intensive research on non-coding RNA, there are also many studies using specially purified small RNA as a template for reverse transcription.
2. Reverse transcriptase (reverse transcriptase): commonly used reverse transcriptases are murine leukemia virus (MMLV) reverse transcriptase and Avian Myeloblastosis Virus (AMV) reverse transcriptase.
3. Primer (primer): primers commonly used for reverse transcription are Oligo-dT and random hexamer primers. Sometimes, specific primers are also used to improve specificity.
4. Reaction buffer: the main components include magnesium salt, potassium salt, buffer component (such as Tris-HCl, etc.) and dNTP, etc.
In addition to the above-mentioned components, an appropriate amount of water is added to prepare a reverse transcription reaction system, and the reaction system is adjusted to a desired volume. It can be said that the composition of the reaction system for reverse transcription is relatively complicated. If several templates are used for reverse transcription at the same time, in order to make the reaction systems as consistent as possible, several components except the templates are mixed into a reverse transcription premix according to a certain proportion, then the prepared premix is divided into several parts, and then the parts are added into the templates respectively. This process requires a certain calculation and is prone to errors in the formulation process by missing the addition of a component.
After reverse transcription to obtain cDNA, the cDNA is often detected using real-time quantitative PCR methods.
Real-time quantitative PCR reactions are generally carried out using 384-well plates or 96-well plates, so that several tens to several hundreds of PCR reactions can be simultaneously carried out to detect a plurality of samples. Each reaction system comprises several components:
1. template: typically cDNA reverse transcribed from mRNA or total RNA.
2. Primers (primers) and probes (probe): the primer is an oligonucleotide fragment which is synthesized according to a template sequence to be detected and can be specifically combined with a certain section of the template, and the primer is divided into a forward primer and a reverse primer, and the length of the primer is generally 15-25 bases. The probe used in the real-time quantitative PCR is generally a fluorescence-labeled probe, and is used for real-time quantitative PCR detection by a probe method. The probe is designed between two primers, and the length of the probe is slightly longer than that of the primers (generally 20-35 bases) so as to ensure that the probe can be bonded on the template before the primers are annealed.
PCR reaction premix (master mix) most of the currently commercially available real-time quantitative PCR reagents are provided in the form of 2 × premix, which includes all components required for PCR reaction except for templates and primers.
In addition to the above-mentioned components, a proper amount of water is added to prepare the reaction system, and the reaction system is adjusted to a desired volume. Thus, 3-4 components are required to be mixed in one reaction system, and hundreds of thousands of pipetting operations are required to complete the preparation of all reaction systems of one 96-well plate or 384-well plate, so that the workload is large. Since each reaction component is in a small amount and is a colorless and transparent solution, the laboratory worker is easy to have repeated loading or missing loading errors in the operation, which results in the failure of the detection.
Therefore, in the reverse transcription-real-time quantitative PCR detection process, the preparation of the reverse transcription and real-time quantitative PCR reaction systems has the following difficulties:
the reverse transcription reaction system has complex composition, the preparation usually needs to be calculated in advance, and the error of adding a certain component is easy to occur in the preparation process. Repeated loading or missing loading errors can easily occur in the preparation of the real-time quantitative PCR reaction system.
To avoid these errors, a variety of real-time quantitative PCR reagents containing dyes are available to the experimenter. However, these dyes are usually only pre-mixed in the PCR reaction premix to help the experimenter confirm whether the PCR reaction premix has been added. This is far from sufficient for the three or four components of each reaction system. Moreover, the problem of the preparation of the reverse transcription reaction system is not solved effectively.
The color reverse transcription premixed solution provided by the invention contains all components of the reverse transcription reaction except the template, the complex operation of mixing a plurality of components (reverse transcriptase, primers and reaction buffer solution) is avoided, the reverse transcription reaction can be conveniently carried out by using the color reverse transcription premixed solution, and the cDNA can be obtained by adding the template and water and reacting at 42 ℃; in addition, the reverse transcription premix also contains dye, and when a reverse transcription system is prepared, whether the reverse transcription premix is added into the system can be confirmed through color, so that the possibility of error occurrence is reduced. The cDNA obtained by reverse transcription also exhibited vivid color. The cDNA is used as a real-time quantitative PCR template, so that an experimenter can be helped to confirm whether the template is correctly added; meanwhile, the method is matched with the real-time quantitative PCR reaction premixed solution containing the dye for use, so that the possibility of error generation during the preparation of a reaction system can be further reduced.
Disclosure of Invention
When reverse transcription-real-time quantitative PCR detection is carried out, errors are easy to occur in the preparation stages of reverse transcription and PCR reaction systems. The invention aims to help the experimenter reduce the possibility of error in the preparation of a reaction system. The implementation mode is to provide a color reverse transcription premix liquid: when preparing a reverse transcription system, only a template and water are added, and the cDNA can be obtained by reaction at 42 ℃; meanwhile, whether the reverse transcription premix is added into the system or not can be confirmed through color. The cDNA obtained using this colored reverse transcription premix also exhibited a vivid color. An experimenter can easily confirm whether template cDNA is added into a PCR reaction system or not through the color of the solution, so that the wrong sample addition is avoided. Therefore, the whole process of reverse transcription-real-time quantitative PCR tracking is realized, and the possibility of error occurrence can be greatly reduced.
Methods for adding dyes to pre-mixes for real-time quantitative PCR reactions have been reported. As mentioned in, for example, the patent application "Method of preparing reaction mixture and related products" (publication No. WO2011039425A 1), a certain concentration of xylene blue FF may be added to the PCR reaction premix, and quinoline yellow may be added to the template or primer. Thus, when a reaction system is prepared, the solution only added with the template or the primer is yellow, the solution only added with the PCR reaction premixed solution is blue, and the solution and the PCR reaction premixed solution are both green. Thus, the reaction solution to which different components are added can be distinguished according to color. However, this method adds a reagent (quinoline yellow, which needs to be added to a template or a primer by an experimenter) in a reaction system, actually makes the preparation process of the reaction system more complicated, and does not solve the problem well or the problem of the preparation of a reverse transcription system.
The invention provides a color reverse transcription premix liquid. The premix contains all components of the reverse transcription reaction except the template, so that the complicated operation of mixing a plurality of components (reverse transcriptase, primers and reaction buffer) is avoided; in addition, the reverse transcription premix also contains dye. The color reverse transcription premixed solution can be used for conveniently carrying out reverse transcription reaction, and cDNA can be obtained by adding a template and water and reacting at 42 ℃. When a reverse transcription system is prepared, whether reverse transcription premix liquid is added into the system or not can be confirmed through color, and the possibility of error occurrence is reduced. The cDNA obtained by reverse transcription also exhibited vivid color. The cDNA is used as a real-time quantitative PCR template, and can help an experimenter to confirm whether the template is added correctly.
Detailed Description
The following embodiments are provided to illustrate specific embodiments of the present invention:
EXAMPLE 1 preparation of a premix for reverse transcription
The components necessary for reverse transcription are: reverse transcriptase (MMLV or AMV reverse transcriptase), magnesium salts (e.g., magnesium chloride, magnesium sulfate, etc.), potassium salts (potassium chloride, potassium sulfate, etc.), buffer components (e.g., Tris-HCl, Bis-Tris, HEPES, Tricine, TAPS, etc.), dNTPs (including dATP, dTTP, dCTP and dGTP), and primers (e.g., Oligo-dT and random hexamer primers), etc. In addition to optimizing the concentration of these essential components, the preparation of the premix for reverse transcription requires the addition and optimization of other components, such as one or more of RNase Inhibitor (RI), glycerol, DTT, and surfactants (such as Triton, Tween, etc.), in order to ensure the stability and reactivity of the premix.
Through trials, an optimized reverse transcription premix formula was obtained as follows:
| components | Concentration of |
| Tris-HCl（pH8.5） | 100 mmol/L |
| MgCl2 | 25 mmol/L |
| KCl | 250 mmol/L |
| DTT | 25 mmol/L |
| dATP | 5 mmol/L |
| dTTP | 5 mmol/L |
| dCTP | 5 mmol/L |
| dGTP | 5 mmol/L |
| Oligo-dT | 0.01 mmol/L |
| Tween 20 | 0.5% |
| RI | 5 U/μL |
| Reverse transcriptase | 50 U/μL |
| Glycerol | 40% |
The reverse transcription premix can be stably stored at-20 ℃ for 2 years.
EXAMPLE 2 preparation of colored reverse transcription premix
Although there are many dyes commonly used in biological experiments, they are not necessarily suitable for the preparation of a color reverse transcription premix. Since the dye added to the premix is present throughout the reverse transcription-real-time quantitative PCR experiment, several factors are considered: firstly, the existence of dye can not interfere the reverse transcription reaction and can not influence the activity and stability of reverse transcriptase; secondly, in general conditions, cDNA obtained by reverse transcription can be used as a template of real-time quantitative PCR after being diluted, so that the color of the dye is bright enough, otherwise, whether the cDNA template is added or not can not be easily seen when a real-time quantitative PCR reaction system is prepared; finally, the presence of the dye does not interfere with the normal performance of the PCR reaction, nor does it interfere with the signal detection of real-time quantitative PCR. Because of the constraints of the above factors, a large amount of experimental screening and verification are required, and suitable dyes are selected and the concentration is optimized.
Several dyes have been found to be useful in attempts: phenol red, neutral red, cresol red, crystal violet, and xylene blue FF. These dyes may be used alone or in combination.
0.02-20 g of dye was weighed and dissolved in 1L of PCR-grade double distilled water. The solution was filtered through a 0.22 μm filter for further use.
When preparing the reverse transcription premix, adding a dye solution except the components in the embodiment 1 to ensure that the concentration of the dye is 0.001-1 g/L, and uniformly mixing to obtain the color reverse transcription premix. The color reverse transcription premixed liquid can be red, blue or purple due to different added dyes, and the shade of the color is related to the concentration of the added dyes. The prepared color reverse transcription premixed solution can be stably stored for 2 years at the temperature of minus 20 ℃.
Example 3 use of colored reverse transcription premix
Carrying out reverse transcription by taking human 293T cell total RNA as a template:
1. RNA concentration was determined by taking 1. mu.g of RNA and adding water to 16. mu.L.
2. Adding 4 mu L of colored reverse transcription premix, blowing, uniformly mixing, centrifuging for a short time, and collecting the liquid to the bottom of the tube.
And incubating for 15-60 minutes at 3.42 ℃ to obtain the cDNA.
As can be seen from the above steps, with the colored reverse transcription premix, the experimenter need only calculate the volumes of the RNA template and water required for the experiment, and then add the fixed volume of the colored reverse transcription premix. The method does not need to mix components such as reverse transcriptase, primers, reaction buffer solution and the like in sequence besides an RNA template and water like the traditional reverse transcription method, and naturally does not need to calculate the volume of the components, thereby greatly simplifying the experimental operation. Furthermore, the solution is colorless before the addition of the colored reverse transcription premix, and is brightly colored red (or blue, purple, depending on the dye in the colored reverse transcription premix) after the addition of the colored reverse transcription premix, and the experimenter can easily determine whether the reverse transcription premix has been added. The cDNA obtained by reverse transcription can be used immediately or stored at-20 ℃ for later use.
Real-time quantitative PCR reactions (prepared on 8-tube, 96-well or 384-well plates):
1. and diluting the cDNA obtained by reverse transcription by 5 times with water, and adding 2-8 mu L of cDNA into each reaction system. Because the cDNA is red (or blue, purple), the experimenter can easily determine whether or not the cDNA has been added.
2. Add 10. mu.L of 2 × real-time quantitative PCR mix, if the real-time quantitative PCR mix is colorless, the solution color in the reaction tube or well is seen to be lighter (because the dye in the cDNA is diluted), and if the real-time quantitative PCR mix also contains dye, the solution color is seen to change.
3. Adding primers and probes with proper concentration (generally, the concentration is in the range of 0.1-1 mu mol/L), adding water to 20 mu L, mixing uniformly, centrifuging for a short time, and collecting the liquid to the bottom of a tube or a hole.
It should be noted that the above-mentioned sample application step is not fixed, but can be adjusted. For example, primers and probes can be added first, followed by cDNA and real-time quantitative PCR mix. The experimenter can flexibly adjust the sample adding sequence according to the actual situation, and the judgment method has no great change.
After the reaction system is prepared, the program is set according to the operation instruction of the real-time quantitative PCR instrument, and the reaction system is put into a reaction tube or a pore plate for detection.
As can be seen from the above description, the use of the colored pre-mixed reverse transcription solution of the present invention can greatly simplify the preparation steps of the reverse transcription reaction system, track the preparation process of the reverse transcription and real-time quantitative PCR reaction system in real time, and determine which components have been added, thereby greatly reducing the possibility of experimental error and improving the experimental detection efficiency.
Claims (9)
1. The color reverse transcription premix comprises the following components: reverse transcriptase, magnesium salt, potassium salt, buffer component, dNTP, primer and dye.
2. The color reverse transcription premix solution of claim 1 wherein the dye is one or more of phenol red, neutral red, cresol red, crystal violet and xylene green FF.
3. The colored reverse transcription premix according to claim 1, wherein the dye concentration is 0.001-1 g/L.
4. The colored reverse transcription premix according to claim 1, further comprising one or more of an rnase inhibitor, glycerol, DTT and a surfactant.
5. The colored reverse transcriptase premix of claim 1 wherein the reverse transcriptase is selected from the group consisting of MMLV reverse transcriptase and AMV reverse transcriptase.
6. The colored reverse transcription premix according to claim 1 wherein the magnesium salt is selected from the group consisting of magnesium chloride and magnesium sulfate.
7. The colored reverse transcription premix according to claim 1 wherein the potassium salt is selected from the group consisting of potassium chloride and potassium sulfate.
8. The color reverse transcription premix according to claim 1 wherein the buffer component is selected from the group consisting of Tris-HCl, Bis-Tris, HEPES, Tricine and TAPS.
9. The colored reverse transcription premix of claim 1 wherein the primer is selected from Oligo-dT and random hexamer primers.
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Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
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| US20150104797A1 (en) * | 2013-10-10 | 2015-04-16 | Bio-Rad Laboratories, Inc. | Composite visible colorant and method for quantitative amplification |
| US20160097086A1 (en) * | 2014-10-03 | 2016-04-07 | Jun Euihum Lee | Compositions and Methods for RT-PCR |
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