CN110055316A - It is a kind of for detect Microrna by terminal deoxynucleotidyl transferase mediate reverse transcription PCR method - Google Patents
It is a kind of for detect Microrna by terminal deoxynucleotidyl transferase mediate reverse transcription PCR method Download PDFInfo
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- C—CHEMISTRY; METALLURGY
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- 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 discloses a kind of for detecting the reverse transcription PCR method of Microrna mediated by terminal deoxynucleotidyl transferase, and especially a kind of reverse transcription PCR detection method of hepatic injury microRNA marker miR-122 belongs to field of biotechnology.Specifically, this method extends the function of single stranded DNA using TdT catalysis RNA, and miR-122 is extended；Then with universal primer reverse transcription is carried out to extension products and with real-time quantitative PCR rapid amplifying, has reached quantitative detection sensitive and special to trace miRNA in pure liquid-phase system.There is this method good specificity, sensitivity and anti-interference ability, the miRNA detection method to establish sensitive and special to provide new direction.
Description
Technical field
The invention discloses a kind of for detecting the reverse transcription of Microrna mediated by terminal deoxyribotide transferase
PCR method belongs to field of biotechnology.
Background technique
In recent years, many Micrornas (microRNAs, miRNAs) have become the potential swollen of early diagnosis of cancer
Tumor markers.In known miRNAs, have it is 50% related with a variety of human tumors, the unconventionality expression of these miRNAs and tumour
Occurrence and development have close relationship.Wherein, 2015 liver cancer (hepatocellular carcinoma, HCC) the whole world about
There are 850,000 new cases and 810,000 lethal cases, disease incidence and lethality all rank among the best.
Since the quantity growth of current China cancer patient is very fast, the science of cancer is prevented, is examined in early days
It is disconnected, timely treatment and monitoring after operation, it has also become the research topic being of great significance and direction.Many biological studies are
It confirms, realizes early diagnosis to cancer, can effectively improve the survival rate of cancer, it can be with by the Sensitive Detection to miRNA
The generation of tumour, development, transfer and prognosis are played an important role.Example miRNA to be checked is miRNA- in the present invention
122, it is molecular biology marker important in the diseases such as liver cancer, hepatic injury.
Traditional detection method mainly includes Northern blotting, microarray and reverse transcriptase real-time quantitative PCR (RT-
PCR).In these methods, Real-time quantitative PCR with respect to easy to operate, sensitivity and specificity for other two kinds of technologies compared with
It is good.However, reverse transcription is fixed in real time because miRNA has the characteristics that sequence is short, expression quantity is low and stability is relatively poor
Amount round pcr usually requires complicated design of primers or Modify to primer, this is unfavorable for the sensitive and general detection of miRNA
's.
In the present invention, a kind of reverse transcription PCR method mediated by terminal deoxyribotide transferase (TdT) is devised.
This method extends the function of single stranded DNA using TdT catalysis RNA, and miRNA is extended；Then with universal primer to extension products
It carries out reverse transcription and with real-time quantitative PCR rapid amplifying, has reached the quantitative detection to trace miRNA.Meanwhile experimental result is demonstrate,proved
The real program has good specificity, sensitivity and anti-interference ability, and for it, further application provides scientific basis.
Summary of the invention
The purpose of the present invention is to provide it is a kind of for detect Microrna by terminal deoxyribotide transferase (TdT)
The reverse transcription PCR method of mediation.
Detection method can be divided into three steps.The first step is that the short nucleic acid that TdT enzyme mediates extends: in free nucleic acid 3 '
In the presence of terminal hydroxy group, TdT enzyme can not catalytic deoxidation ribose triphosphoric acid polymerization reaction；In the presence of having miRNA, TdT enzyme can be
A DNA chain is extended at its 3 ' end, to solve the problems, such as that target is too short.In view of the extension of TdT is not depend on template
, when, there are when a variety of dNTPs, extension sequence can have randomness in solution, so for the ease of the spy of primer when reverse transcription
The opposite sex combines, and a kind of dNTP is only added in we in the solution.Second step is reverse transcription reaction: reverse transcriptase primer (primer I) can be special
The opposite sex combines the obtained long-chain of first step extension, then obtained under the mediation of reverse transcriptase one it is corresponding with target
CDNA lengthens chain.5 ' ends of this chain can have one section of sequence complementary with next step universal primer because of primer I, after this is
It carries out efficient amplification and has laid solid foundation.Third step is real-time fluorescence quantitative PCR reaction: this step is to lengthen chain with cDNA to be
Template, binding specificity primer (primer II) and universal primer (primer III) carry out the PCR amplification of Taq enzyme mediation.It was expanding
Cheng Zhong, the dyestuff in system can specifically bind DNA double chain and issue fluorescence signal, can be grasped indirectly by capturing the signal
The content of amplified production in system.Finally, by software analysis meter calculate system fluorescence intensity reach threshold value needed for after PCR
Cycle-index (i.e. Ct value).
The invention provides the following technical scheme:
It is a kind of for detect Microrna by terminal deoxynucleotidyl transferase mediate reverse transcription PCR method, detection
Method includes the following steps:
(1) terminal deoxynucleotidyl transferase mediates the 3 ' of Microrna miR-122 using deoxynucleoside triphosphate as substrate
C-terminal extends, wherein the sequence of the miR-122 is as shown in SEQ ID No.5；
(2) reverse transcriptase mediates extension products to carry out reverse transcription in the case where reverse transcriptase primer I participates in, and is used for reverse transcription
I sequence of primer of reaction is as shown in SEQ ID No.1；
(3) reverse transcription product carries out PCR amplification with specific primer II, universal primer III under archaeal dna polymerase mediation, real
The detection of existing Microrna miR-122 for mediating the specific primer II of pcr amplification reaction as shown in SEQ ID No.2, is led to
With primer III as shown in SEQ ID No.3.
In the reverse transcription PCR method mediated by terminal deoxynucleotidyl transferase for detecting Microrna, end
Transferase is using RNA as primer extend.
In the reverse transcription PCR method mediated by terminal deoxynucleotidyl transferase for detecting Microrna, end
Deoxynucleotidyl transferase mediate Microrna 3 ' C-terminals extend the following steps are included:
It prepares and contains 250 μM of CoCl2(offer of terminal deoxynucleotidyl transferase kit), 1 × end deoxynucleotide
The reaction system of enzyme reaction buffer solution (offer of terminal deoxynucleotidyl transferase kit) and 1 μM of -1mM dTTP, Xiang Ti are provided
Microrna and 10U terminal deoxynucleotidyl transferase to be measured, 37 DEG C of reaction 30-60min of constant temperature, 85 DEG C of inactivations are added in system
25min。
In the reverse transcription PCR method mediated by terminal deoxynucleotidyl transferase for detecting Microrna, reversion
Record enzyme mediate primer I participate in reverse transcription the following steps are included:
Prepare the upper step reaction product containing 10 times of dilution, 1nM-10 μM of primer I, 1 × reverse transcription buffer (contain Mg2+
And dNTP, provided with Reverse Transcriptase Reagents kit) and reverse transcriptase Mix I (containing reverse transcriptase and RNase inhibitor, with reversion
Enzyme reagent kit is recorded to provide) reaction system, then 37 DEG C of reaction 15-30min of constant temperature, then 85 DEG C of inactivation 5s.
In the reverse transcription PCR method mediated by terminal deoxynucleotidyl transferase for detecting Microrna, primer
I design: primer I includes that one section of region complementary with the sequence that terminal deoxynucleotidyl transferase extends and one section are used for
The primer binding zone of amplification.
In the reverse transcription PCR method mediated by terminal deoxynucleotidyl transferase for detecting Microrna, DNA
The PCR amplification step concrete operations that polymerase-mediated primer II, III participates in are as follows:
Reaction carries out in 96PCR pipe, first in hole in pipe configuration containing 10 times of dilution reverse transcription product,
The reaction system of 250nMPrimer II, 250nM Primer III and 1 × SYBR Premix ExTMTaq II, then put plate
EnterPCR reaction is carried out in 480Instrument II (Roche) fluorescence quantitative PCR instrument.Program is 95 DEG C and adds
Hot 30s, then circulation carries out 95 DEG C of heating 5s, 63 DEG C of extension 30s, terminates reaction after circulation 50 times.
In the reverse transcription PCR method mediated by terminal deoxynucleotidyl transferase for detecting Microrna, PCR
The design of amplimer II, III: the sequence of primer II is the DNA sequence identical with target Microrna other than base type
Column；Primer III is consistent with the primer binding zone sequence in primer I for amplification.
The reverse transcription PCR method mediated by terminal deoxynucleotidyl transferase for detecting Microrna is for liver
Organize the detection of miR-122 in microRNA extracting solution.
The beneficial effects of the present invention are:
First in suitable buffer, extends a DNA " tail " to 3 ' C-terminals of Microrna with TdT, make
Obtain the region that target generates one section and reverse transcriptase primer combination while elongated.Then, it is obtained by reverse transcription step pure
DNA chain is to carry out the PCR amplification based on Taq enzyme.Finally, passing through work with the content of real-time quantitative PCR detection reverse transcription product
Make curve and finally determines Microrna content in sample.
It is sensitive: to compare other methods, TdT extension method in conjunction with reverse transcription technology, is detected the line of miR-122 by this method
Property range be 100fM~1nM.
It is special: to ensure that specificity with the consistent specific primer of target miRNA sequence, detectable concentration is in 10pM level
When, the program can be effectively by there are the segments of base difference to distinguish with target.
Probe versatility: specific primer need to only be become when change detection target miRNA consistent with target miRNA sequence
DNA sequence dna, without converting other primers.
The present invention provides the reverse transcription PCR methods mediated by terminal deoxynucleotidyl transferase.And utilize the above method
MiRNA is quantified, a series of excellent technical effects are achieved:
Specificity analysis finds detectable concentration in 10pM level, and the program can be effectively by there are single bases with target
The segment of difference distinguishes, and illustrates that its detection specificity is good.Then we learn in the drawing process of working curve, this
Illustrate that its detection sensitivity is very high.Finally, we have carried out Standard entertion experiment with this method, it was demonstrated that this method has one
Fixed anti-interference ability and has and be applied to actually detected potentiality.
Detailed description of the invention
The invention will be further described with specific embodiment with reference to the accompanying drawings of the specification.
The reverse transcription method basic procedure schematic diagram that Fig. 1 TdT is mediated；
Stage difference dTTP Concentration Testing DNA122 is extended based on TdT in Fig. 2 present invention；
I Concentration Testing DNA122 of reverse transcription stage different primers is based in Fig. 3 present invention；
MiR-122 detection effect of Fig. 4 present invention to various concentration；
The working curve of Fig. 5 this method；
Fig. 6 specificity experiments；
MiR-122 standard items are added into actual sample and detect by Fig. 7；
Specific embodiment:
Below to specific embodiments of the present invention be described in detail in order to those skilled in the art understand that the present invention, but
And the invention is not limited in any way.For those skilled in the art, present inventive concept is not being departed from
Under the premise of, improvements and modifications can be made, these improvements and modifications are within protection scope of the present invention.
Key instrument and reagent: PrimeScriptTMReverse transcription reagent box, Premix ExTMTaqⅡ(Tli
RNaseH Plus), dTTP be purchased from precious bioengineering (Dalian) Co., Ltd；MiRcute miRNA separating kit is purchased from Tiangeng
Biochemical technology (Beijing) Co., Ltd；Terminal deoxynucleotidyl transferase, reaction buffer and CoCl2(2.5mM) is purchased from knob English
Human relations biotechnology (Beijing) Co., Ltd；Nucleic acid particular sequence used in the present invention is listed in sequence table, synthesis is raw in raw work
Object engineering (Shanghai) limited liability company.
The detection side of hepatic injury marker miR-122 is used for by the reverse transcription PCR that terminal deoxynucleotidyl transferase mediates
Method, as shown in Figure 1, the detection method includes:
Terminal deoxynucleotidyl transferase mediates the 3 ' C-terminals of miR-122 to prolong using deoxynucleoside triphosphate as substrate
It stretches, reverse transcriptase mediates extension products to carry out reverse transcription in the case where reverse transcriptase primer I participates in, and reverse transcription product is poly- in DNA
Synthase mediates lower and specific primer II, universal primer III to carry out PCR amplification, realizes the detection of miR-122；
Wherein, the sequence of the miR-122 is as shown in SEQ ID No.5；I sequence of primer for reverse transcription reaction is such as
Shown in SEQ ID No.1；For mediating the specific primer II of pcr amplification reaction as shown in SEQ ID No.2, universal primer III
As shown in SEQ ID No.3.
The reverse transcription PCR that the terminal deoxynucleotidyl transferase mediates is used for the detection of hepatic injury marker miR-122
In method, terminal enzyme (DNA) is using RNA as primer extend.
Terminal deoxynucleotidyl transferase mediate Microrna 3 ' C-terminals extend the following steps are included:
It prepares and contains 250 μM of CoCl2(offer of terminal deoxynucleotidyl transferase kit), 1 × end deoxynucleotide
The reaction system of enzyme reaction buffer solution (offer of terminal deoxynucleotidyl transferase kit) and 1 μM of -1mM dTTP, Xiang Ti are provided
Microrna and 10U terminal deoxynucleotidyl transferase to be measured, 37 DEG C of reaction 30-60min of constant temperature, 85 DEG C of inactivations are added in system
25min。
The reverse transcription PCR that the terminal deoxynucleotidyl transferase mediates is used for the detection of hepatic injury marker miR-122
In method, reverse transcriptase mediate primer I participate in reverse transcription the following steps are included:
Prepare the upper step reaction product containing 10 times of dilution, 1nM-10 μM of primer I, 1 × reverse transcription buffer (contain Mg2+
And dNTP, provided with Reverse Transcriptase Reagents kit) and reverse transcriptase Mix I (containing reverse transcriptase and RNase inhibitor, with reversion
Enzyme reagent kit is recorded to provide) reaction system, then 37 DEG C of reaction 15min of constant temperature, then 85 DEG C of inactivation 5s.
The reverse transcription PCR that the terminal deoxynucleotidyl transferase mediates is used for the detection of hepatic injury marker miR-122
In method, primer I includes that one section of region complementary with the sequence that terminal deoxynucleotidyl transferase extends and one section are used for
The primer binding zone of amplification.
The reverse transcription PCR that the terminal deoxynucleotidyl transferase mediates is used for the detection of hepatic injury marker miR-122
In method, the PCR amplification step concrete operations that the primer II, III that archaeal dna polymerase mediates participates in are as follows:
Reaction carries out in 96PCR pipe, first reverse transcription product of the configuration containing 10 times of dilution, 250nM in the hole in pipe
Plate, is then put by the reaction system of primer II, 250nM primer III and 1 × SYBR Premix ExTMTaq IIPCR reaction is carried out in 480Instrument II (Roche) fluorescence quantitative PCR instrument.Program is 95 DEG C of heating
30s, then circulation carries out 95 DEG C of heating 5s, 63 DEG C of extension 30s, terminates reaction after circulation 50 times.
The reverse transcription PCR that the terminal deoxynucleotidyl transferase mediates is used for the detection of hepatic injury marker miR-122
The sequence of primer II is the DNA sequence dna identical with target Microrna other than base type in method；Primer III and primer
Primer binding zone sequence in I for amplification is consistent.
The reverse transcription PCR that the terminal deoxynucleotidyl transferase mediates is used for the detection of hepatic injury marker miR-122
In method, the detecting step of miR-122 is as follows in hepatic tissue microRNA extracting solution:
(1) the extraction separation that separating kit carries out miRNA in murine liver tissue is extracted using miRcute miRNA.It presses
Operating procedure is tested on specification, finally by NanoDropTMDetect the concentration of the miRNA isolated.
(2) obtained murine liver tissue extracting solution is diluted to 2.5ng/ μ L；Compound concentration is the miR- of 1nM and 10nM
122 solution.
(3) extend step in TdT, system includes 5-10ng murine liver tissue miRNA extracting solution, various concentration miR-122
(final concentration is respectively 100pM-10nM), 1 μM of -1mM dTTP, 250 μM of CoCl2, 1 × TdT reaction buffer and 10U TdT
The reaction system of enzyme, then 37 DEG C of reaction 30-60min of constant temperature, then 85 DEG C of inactivation 25min.
(4) the upper step reaction product containing 10 times of dilution, 1nM-10 μM of primer I, 1 × reverse transcription reverse transcription step: are prepared
The reaction system of buffer and reverse transcriptase Mix I, then 37 DEG C of reaction 15-30min of constant temperature, then 85 DEG C of inactivation 5s.
(5) real-time fluorescence quantitative PCR: reaction carries out on 96PCR plate, and first configuration contains the reversion for diluting 10 times in hole
The reaction system for recording product, 250nM primer II, 250nM primer III and 1 × SYBR Premix ExTMTaq II, then by plate
It is put into480Instrument II carries out PCR reaction.Program is first 95 DEG C of heating 30s, and then circulation carries out
95 DEG C are heated 5s and 63 DEG C of extension 30s, terminate reaction after 50 circulations.It utilizes480Instrument II into
Row data record.
Embodiment 1:
(1) TdT extends step: first preparing and contains 1 μM of dTTP, 250 μM of CoCl2, 10pM DNA-122 (sequence such as SEQ
Shown in ID No.4), the reaction system of 1 × TdT reaction buffer and 10U TdT enzyme, then 37 DEG C of reaction 30min of constant temperature, connect
85 DEG C of inactivation 25min.
(2) the upper step reaction product containing 10 times of dilution, (the sequence such as SEQ ID of 100nM primer I reverse transcription step: are prepared
Shown in No.1), the reaction system of 1 × reverse transcription buffer and reverse transcriptase Mix I, then 37 DEG C of reaction 15min of constant temperature, connect
85 DEG C of inactivation 5s.
(3) real-time fluorescence quantitative PCR: reaction carries out on 96PCR plate, and first configuration contains the reversion for diluting 10 times in hole
Record product, 250nM primer II (sequence is as shown in SEQ ID No.2), 250nM primer III (sequence is as shown in SEQ ID No.3)
And the reaction system of 1 × SYBR Premix ExTMTaq II, then plate is put into480Instrument
II carries out PCR reaction.Program is first 95 DEG C of heating 30s, and then circulation carries out 5s and 63 DEG C of extension 30s of 95 DEG C of heating, and 50 are followed
Reaction is terminated after ring.It utilizes480Instrument II carries out data record.
As a result: it is 10pM that experiment, which has been investigated by model of DNA-122 in DNA-122 content, dTTP concentration in extension system
Influence to detection effect, experimental result are presented in Fig. 2.As shown in Fig. 2, Δ Ct value is when dTTP concentration is 1 μM in system
20.03, larger but not up to highest.
Embodiment 2:
(1) TdT extends step: first preparing and contains 10 μM of dTTP, 250 μM of CoCl2, 10pM DNA-122,1 × TdT it is anti-
The reaction system of buffer and 10U TdT enzyme is answered, then 37 DEG C of reaction 30min of constant temperature, then 85 DEG C of inactivation 25min.
(2) the upper step reaction product containing 10 times of dilution, 100nM primer I, 1 × reverse transcription buffering reverse transcription step: are prepared
The reaction system of liquid and reverse transcriptase Mix I, then 37 DEG C of reaction 15min of constant temperature, then 85 DEG C of inactivation 5s.
(3) real-time fluorescence quantitative PCR: reaction carries out on 96PCR plate, and first configuration contains the reversion for diluting 10 times in hole
The reaction system for recording product, 250nM primer II, 250nM primer III and 1 × SYBR Premix ExTMTaq II, then by plate
It is put into480Instrument II carries out PCR reaction.Program is first 95 DEG C of heating 30s, and then circulation carries out
95 DEG C are heated 5s and 63 DEG C of extension 30s, terminate reaction after 50 circulations.It utilizes480Instrument II into
Row data record.
As a result: experiment has been investigated when DNA-122 content is 10pM, shadow of the dTTP concentration to detection effect in extension system
It rings, experimental result is presented in Fig. 2.As shown in Fig. 2, Δ Ct value reaches peak when dTTP concentration is 10 μM in system
20.12, it is seen that when dTTP concentration is 1 μM of -1mM in system, Δ Ct value is larger and relatively stable.
Embodiment 3:
(1) TdT extends step: first preparing and contains 10 μM of dTTP, 250 μM of CoCl2, 10pM DNA-122,1 × TdT it is anti-
The reaction system of buffer and 10U TdT enzyme is answered, then 37 DEG C of reaction 30min of constant temperature, then 85 DEG C of inactivation 25min.
(2) the upper step reaction product containing 10 times of dilution, 100nM primer I, 1 × reverse transcription buffering reverse transcription step: are prepared
The reaction system of liquid and reverse transcriptase Mix I, then 37 DEG C of reaction 15min of constant temperature, then 85 DEG C of inactivation 5s.
(3) real-time fluorescence quantitative PCR: reaction carries out on 96PCR plate, and first configuration contains the reversion for diluting 10 times in hole
The reaction system for recording product, 250nM primer II, 250nM primer III and 1 × SYBR Premix ExTMTaq II, then by plate
It is put into480Instrument II carries out PCR reaction.Program is first 95 DEG C of heating 30s, and then circulation carries out
95 DEG C are heated 5s and 63 DEG C of extension 30s, terminate reaction after 50 circulations.It utilizes480Instrument II into
Row data record.
As a result: experiment has been investigated when DNA-122 content is 10pM, shadow of the dTTP concentration to detection effect in extension system
It rings, experimental result is presented in Fig. 2.As shown in Fig. 2, Δ Ct value is 20.13, close to most when dTTP concentration is 100 μM in system
High level, it is seen that when dTTP concentration is 1 μM of -1mM in system, Δ Ct value is larger and relatively stable.
Embodiment 4:
(1) TdT extends step: first preparing and contains 100 μM of dTTP, 250 μM of CoCl2, 10pM DNA-122,1 × TdT it is anti-
The reaction system of buffer and 10U TdT enzyme is answered, then 37 DEG C of reaction 30min of constant temperature, then 85 DEG C of inactivation 25min.
(2) the upper step reaction product containing 10 times of dilution, 100nM primer I, 1 × reverse transcription buffering reverse transcription step: are prepared
The reaction system of liquid and reverse transcriptase Mix I, then 37 DEG C of reaction 15min of constant temperature, then 85 DEG C of inactivation 5s.
(3) real-time fluorescence quantitative PCR: reaction carries out on 96PCR plate, and first configuration contains the reversion for diluting 10 times in hole
The reaction system for recording product, 250nM primer II, 250nM primer III and 1 × SYBR Premix ExTMTaq II, then by plate
It is put into480Instrument II carries out PCR reaction.Program is first 95 DEG C of heating 30s, and then circulation carries out
95 DEG C are heated 5s and 63 DEG C of extension 30s, terminate reaction after 50 circulations.It utilizes480Instrument II into
Row data record.
As a result: experiment has been investigated when DNA-122 content is 10pM, shadow of the dTTP concentration to detection effect in extension system
It rings, experimental result is presented in Fig. 2.As shown in Fig. 2, Δ Ct value is 15.04 when dTTP concentration is 1mM in system, show down
The trend of drop, it is seen that when dTTP concentration is 1 μM of -1mM in system, Δ Ct value is larger and relatively stable, bright more than declining after 1mM
It is aobvious.
Embodiment 5:
(1) TdT extends step: first preparing and contains 1mM dTTP, 250 μM of CoCl2, 10pM DNA-122,1 × TdT reaction
The reaction system of buffer and 10U TdT enzyme, then 37 DEG C of reaction 30min of constant temperature, then 85 DEG C of inactivation 25min.
(2) the upper step reaction product containing 10 times of dilution, 100nM primer I, 1 × reverse transcription buffering reverse transcription step: are prepared
The reaction system of liquid and reverse transcriptase Mix I, then 37 DEG C of reaction 15min of constant temperature, then 85 DEG C of inactivation 5s.
(3) real-time fluorescence quantitative PCR: reaction carries out on 96PCR plate, and first configuration contains the reversion for diluting 10 times in hole
The reaction system for recording product, 250nM primer II, 250nM primer III and 1 × SYBR Premix ExTMTaq II, then by plate
It is put into480Instrument II carries out PCR reaction.Program is first 95 DEG C of heating 30s, and then circulation carries out
95 DEG C are heated 5s and 63 DEG C of extension 30s, terminate reaction after 50 circulations.It utilizes480Instrument II into
Row data record.
As a result: experiment has been investigated when DNA-122 content is 10pM, shadow of the dTTP concentration to detection effect in extension system
It rings, experimental result is presented in Fig. 2.As shown in Fig. 2, Δ Ct value is 0.10 when dTTP concentration is 10mM in system, it cannot be by reality
It tests group to distinguish with control group, it is seen that when dTTP concentration is only 1 μM of -1mM in system, Δ Ct value is larger and relatively stable.
Embodiment 6:
(1) TdT extends step: first preparing and contains 100 μM of dTTP, 250 μM of CoCl2, 10pM DNA-122,1 × TdT it is anti-
The reaction system of buffer and 10U TdT enzyme is answered, then 37 DEG C of reaction 30min of constant temperature, then 85 DEG C of inactivation 25min.
(2) the upper step reaction product containing 10 times of dilution, 1nM primer I, 1 × reverse transcription buffer reverse transcription step: are prepared
And the reaction system of reverse transcriptase Mix I, then 37 DEG C of reaction 15min of constant temperature, then 85 DEG C of inactivation 5s.
(3) real-time fluorescence quantitative PCR: reaction carries out on 96PCR plate, and first configuration contains the reversion for diluting 10 times in hole
The reaction system for recording product, 250nM primer II, 250nM primer III and 1 × SYBR Premix ExTMTaq II, then by plate
It is put into480Instrument II carries out PCR reaction.Program is first 95 DEG C of heating 30s, and then circulation carries out
95 DEG C are heated 5s and 63 DEG C of extension 30s, terminate reaction after 50 circulations.It utilizes480Instrument II into
Row data record.
As a result: when having investigated using extension products progress reverse transcription PCR, I content of primer is to detection effect in system
Influence (experimental result is presented in Fig. 3).As shown in figure 3, Δ Ct value is 20.67 when I concentration of system inner primer is 1nM, it can
It is obvious to distinguish signal and background, it is seen that when I concentration of system inner primer is 1nM-10 μM, to there is detection effect.
Embodiment 7:
(1) TdT extends step: first preparing and contains 100 μM of dTTP, 250 μM of CoCl2, 10pM DNA-122,1 × TdT it is anti-
The reaction system of buffer and 10U TdT enzyme is answered, then 37 DEG C of reaction 30min of constant temperature, then 85 DEG C of inactivation 25min.
(2) the upper step reaction product containing 10 times of dilution, 10nM primer I, 1 × reverse transcription buffering reverse transcription step: are prepared
The reaction system of liquid and reverse transcriptase Mix I, then 37 DEG C of reaction 15min of constant temperature, then 85 DEG C of inactivation 5s.
(3) real-time fluorescence quantitative PCR: reaction carries out on 96PCR plate, and first configuration contains the reversion for diluting 10 times in hole
The reaction system for recording product, 250nM primer II, 250nM primer III and 1 × SYBR Premix ExTMTaq II, then by plate
It is put into480Instrument II carries out PCR reaction.Program is first 95 DEG C of heating 30s, and then circulation carries out
95 DEG C are heated 5s and 63 DEG C of extension 30s, terminate reaction after 50 circulations.It utilizes480Instrument II into
Row data record.
As a result: when experiment has been investigated using extension products progress reverse transcription PCR, I content of primer is to detection in system
The influence of effect (experimental result is presented in Fig. 3).As shown in figure 3, Δ Ct value is when I concentration of system inner primer is 10nM
21.62, can obviously distinguish signal and background has detection effect when I concentration of system inner primer is 1nM-10 μM.
Embodiment 8:
(1) TdT extends step: first preparing anti-containing 250 μM of CoCl2,10pM DNA-122,100 μM of dTTP, 1 × TdT
The reaction system of buffer and 10U TdT enzyme is answered, then 37 DEG C of reaction 30min of constant temperature, then 85 DEG C of inactivation 25min.
(2) the upper step reaction product containing 10 times of dilution, 100nM primer I, 1 × reverse transcription buffering reverse transcription step: are prepared
The reaction system of liquid and reverse transcriptase Mix I, then 37 DEG C of reaction 15min of constant temperature, then 85 DEG C of inactivation 5s.
(3) real-time fluorescence quantitative PCR: reaction carries out on 96PCR plate, and first configuration contains the reversion for diluting 10 times in hole
The reaction system for recording product, 250nM primer II, 250nM primer III and 1 × SYBR Premix ExTMTaq II, then by plate
It is put into480Instrument II carries out PCR reaction.Program is first 95 DEG C of heating 30s, and then circulation carries out
95 DEG C are heated 5s and 63 DEG C of extension 30s, terminate reaction after 50 circulations.It utilizes480Instrument II into
Row data record.
As a result: when experiment has been investigated using extension products progress reverse transcription PCR, I content of primer is to detection in system
The influence of effect (experimental result is presented in Fig. 3).When I concentration of system inner primer is 100nM, Δ Ct value is 21.43, can be obvious
Distinguish signal and background.When I concentration of system inner primer is 1nM-10 μM as shown in Figure 3, there is detection effect.
Embodiment 9:
(1) TdT extends step: first preparing anti-containing 250 μM of CoCl2,10pM DNA-122,100 μM of dTTP, 1 × TdT
The reaction system of buffer and 10U TdT enzyme is answered, then 37 DEG C of reaction 30min of constant temperature, then 85 DEG C of inactivation 25min.
(2) the upper step reaction product containing 10 times of dilution, 1 μM of primer I, 1 × reverse transcription buffer reverse transcription step: are prepared
And the reaction system of reverse transcriptase Mix I, then 37 DEG C of reaction 15min of constant temperature, then 85 DEG C of inactivation 5s.
(3) real-time fluorescence quantitative PCR: reaction carries out on 96PCR plate, and first configuration contains the reversion for diluting 10 times in hole
The reaction system for recording product, 250nM primer II, 250nM primer III and 1 × SYBR Premix ExTMTaq II, then by plate
It is put into480Instrument II carries out PCR reaction.Program is first 95 DEG C of heating 30s, and then circulation carries out
95 DEG C are heated 5s and 63 DEG C of extension 30s, terminate reaction after 50 circulations.It utilizes480Instrument II into
Row data record.
As a result: when experiment has been investigated using extension products progress reverse transcription PCR, I content of primer is to detection in system
The influence of effect (experimental result is presented in Fig. 3).When I concentration of system inner primer is 1 μM, Δ Ct value is 20.63, can obvious area
Sub-signal and background.It can be seen that having detection effect when I concentration of system inner primer is 1nM-10 μM.
Embodiment 10:
(4) TdT extends step: first preparing anti-containing 100 μM of dTTP, 250 μM of CoCl2,10pM DNA-122,1 × TdT
The reaction system of buffer and 10U TdT enzyme is answered, then 37 DEG C of reaction 30min of constant temperature, then 85 DEG C of inactivation 25min.
(5) the upper step reaction product containing 10 times of dilution, 10 μM of primers I, 1 × reverse transcription buffering reverse transcription step: are prepared
The reaction system of liquid and reverse transcriptase Mix I, then 37 DEG C of reaction 15min of constant temperature, then 85 DEG C of inactivation 5s.
(6) real-time fluorescence quantitative PCR: reaction carries out on 96PCR plate, and first configuration contains the reversion for diluting 10 times in hole
The reaction system for recording product, 250nM primer II, 250nM primer III and 1 × SYBR Premix ExTMTaq II, then by plate
It is put into480Instrument II carries out PCR reaction.Program is first 95 DEG C of heating 30s, and then circulation carries out
95 DEG C are heated 5s and 63 DEG C of extension 30s, terminate reaction after 50 circulations.It utilizes480Instrument II into
Row data record.
As a result: when experiment has been investigated using extension products progress reverse transcription PCR, I content of primer is to detection in system
The influence of effect (experimental result is presented in Fig. 3).When I concentration of system inner primer is 10 μM, Δ Ct value is 17.50, can be obvious
Signal and background are distinguished, but numerical value is substantially reduced, detection effect is deteriorated.
Embodiment 11:
(1) compound concentration is distinguished for miR-122 (the sequence such as SEQ ID No.5 institute of 1pM, 10pM, 100pM, 1nM, 10nM
Show) solution.
(2) TdT extend step, system include various concentration miR-122 (final concentration be respectively 100fM, 1pM, 10pM,
100pM and 1nM), 100 μM of dTTP, 250 μM of CoCl2, 1 × TdT reaction buffer and 10U TdT enzyme reaction system, so
37 DEG C of reaction 30min of constant temperature afterwards, then 85 DEG C of inactivation 25min.
(3) the upper step reaction product containing 10 times of dilution, 1 μM of primer I, 1 × reverse transcription buffer reverse transcription step: are prepared
And the reaction system of reverse transcriptase Mix I, then 37 DEG C of reaction 15min of constant temperature, then 85 DEG C of inactivation 5s.
(4) real-time fluorescence quantitative PCR: reaction carries out on 96PCR plate, and first configuration contains the reversion for diluting 10 times in hole
The reaction system for recording product, 250nM primer II, 250nM primer III and 1 × SYBR Premix ExTMTaq II, then by plate
It is put into480Instrument II carries out PCR reaction.Program is first 95 DEG C of heating 30s, and then circulation carries out
95 DEG C are heated 5s and 63 DEG C of extension 30s, terminate reaction after 50 circulations.It utilizes480Instrument II into
Row data record.
As a result: detecting various concentration miR-122 with this method, obtain real-time fluorescence curves figure (Fig. 4), and further draw
Working curve (Fig. 5).Curve linear range 100fM~1nM, regression equation are as follows: Ct value=- 4.46lg (A)-
21.57 (R2=0.99556, Ct value be by software analysis meter calculate system fluorescence intensity reach threshold value needed for after PCR follow
Ring number, A are the substance withdrawl syndrome numerical value of miR-122 in extension system).
Embodiment 12:
(1) compound concentration is 100pM respectively miR-122, Smi-122, Dmi-122, Tmi-122, miR-21, miR-
141 and single-stranded NC solution.TdT is successively carried out later extends T, reverse transcription and real-time fluorescence quantitative PCR.
(2) extend step in TdT, system include different target that final concentration is 10pM (miR-122, Smi-122,
Dmi-122, Tmi-122, miR-21, miR-141 and single-stranded NC), 100 μM of dTTP, 250 μM of CoCl2, 1 × TdT reaction it is slow
The reaction system of fliud flushing and 10U TdT enzyme, then 37 DEG C of reaction 30min of constant temperature, then 85 DEG C of inactivation 25min；Smi-122,
Dmi-122, Tmi-122, miR-21, miR-141 and single-stranded NC sequence such as SEQ6Smi-122, SEQ7Dmi-122,
Shown in the single-stranded negative control of SEQ8Tmi-122, SEQ9miR-21, SEQ10miR-141, SEQ11NC.
(3) the upper step reaction product containing 10 times of dilution, 1 μM of primer I, 1 × reverse transcription buffer reverse transcription step: are prepared
And the reaction system of reverse transcriptase Mix I, then 37 DEG C of reaction 15min of constant temperature, then 85 DEG C of inactivation 5s.
(4) real-time fluorescence quantitative PCR: reaction carries out on 96PCR plate, and first configuration contains the reversion for diluting 10 times in hole
The reaction system for recording product, 250nM primer II, 250nM primer III and 1 × SYBR Premix ExTMTaq II, then by plate
It is put into480Instrument II carries out PCR reaction.Program is first 95 DEG C of heating 30s, and then circulation carries out
95 DEG C are heated 5s and 63 DEG C of extension 30s, terminate reaction after 50 circulations.It utilizes480 Instrument II
Carry out data record.
As a result: detected respectively with this method miR-122, Smi-122 of 10pM, Dmi-122, Tmi-122, miR-21,
MiR-141 and NC (Negative control).As shown in fig. 6, detecting the Δ Ct value of miR-122 when fragment concentrations are identical
It is significantly higher than the Δ Ct value of detection other types RNA segment.Illustrate with the help of specific primer, the program can be realized effectively
To the specific detection of miR-122.
Embodiment 13:
(1) the extraction separation that separating kit carries out miRNA in murine liver tissue is extracted using miRcute miRNA.It presses
Operating procedure is tested on specification, finally by NanoDropTMDetect the concentration of the miRNA isolated.
(2) obtained murine liver tissue miRNA extracting solution is diluted to 2.5ng/ μ L.
(3) compound concentration is the miR-122 solution of 1nM and 10nM.
(4) extend step in TdT, system includes 5ng murine liver tissue miRNA extracting solution, various concentration miR-122 (eventually
Concentration is respectively 100pM and 1nM), 100 μM of dTTP, 250 μM of CoCl2, 1 × TdT reaction buffer and 10U TdT enzyme
Reaction system, then 37 DEG C of reaction 30min of constant temperature, then 85 DEG C of inactivation 25min.
(5) the upper step reaction product containing 10 times of dilution, 1 μM of primer I, 1 × reverse transcription buffer reverse transcription step: are prepared
And the reaction system of reverse transcriptase Mix I, then 37 DEG C of reaction 15min of constant temperature, then 85 DEG C of inactivation 5s.
(6) real-time fluorescence quantitative PCR: reaction carries out on 96PCR plate, and first configuration contains the reversion for diluting 10 times in hole
The reaction system for recording product, 250nM primer II, 250nM primer III and 1 × SYBR Premix ExTMTaq II, then by plate
It is put into480Instrument II carries out PCR reaction.Program is first 95 DEG C of heating 30s, and then circulation carries out
95 DEG C are heated 5s and 63 DEG C of extension 30s, terminate reaction after 50 circulations.It utilizes480 Instrument II
Carry out data record.
As a result:, can be with by the way that miR-122 standard items are added into actual sample and with our new method measurement rate of recovery
Analyze the anti-interference ability of our methods.The Ct value that replication three times obtains is averaged, working curve recurrence side is substituted into
Journey calculates the miR-122 (group1 in Fig. 7) in the sample of 250 μ g/L containing 67.31pM；It is calculated with same procedure to 250
After 50pM miR-122 standard items are added in the sample of μ g/L, mixture miR-122 concentration measurement is 110.88pM (in Fig. 7
Group2), the rate of recovery 87.1%；After 100pM miR-122 standard items are added into same concentrations sample, miR- in mixture
122 concentration become 151.40pM (group3 in Fig. 7), the rate of recovery 84.1%.From the results of view, this method has good anti-dry
Disturb ability.
Specific embodiments of the present invention are illustrated above, it should be noted that the present invention is not limited to above-mentioned reality
Mode is applied, for those skilled in the art, without departing from the principle of the present invention, can also be made
Several improvements and modifications, these improvements and modifications are also considered to be within the protection scope of the present invention.
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Claims (8)
1. a kind of for detecting the reverse transcription PCR method of Microrna mediated by terminal deoxynucleotidyl transferase, feature exists
In detection method includes the following steps:
(1) terminal deoxynucleotidyl transferase mediates the 3 ' hydroxyls of Microrna miR-122 using deoxynucleoside triphosphate as substrate
End extends, wherein the sequence of the miR-122 is as shown in SEQ ID No.5；
(2) reverse transcriptase mediates extension products to carry out reverse transcription in the case where reverse transcriptase primer I participates in, and is used for reverse transcription reaction
I sequence of primer as shown in SEQ ID No.1；
(3) reverse transcription product carries out PCR amplification with specific primer II, universal primer III under archaeal dna polymerase mediation, realizes micro-
The detection of tiny RNA miR-122 for mediating the specific primer II of pcr amplification reaction as shown in SEQ ID No.2, general is drawn
Object III is as shown in SEQ ID No.3.
2. method according to claim 1, which is characterized in that it is described for detect Microrna by end deoxynucleotide
In transferase mediated reverse transcription PCR method, terminal enzyme (DNA) is using RNA as primer extend.
3. method according to claim 1, which is characterized in that it is described for detect Microrna by end deoxynucleotide
In transferase mediated reverse transcription PCR method, 3 ' C-terminals of the Microrna that terminal deoxynucleotidyl transferase mediates extend
The following steps are included:
It prepares and contains 250 μM of CoCl2The transfer of (offer of terminal deoxynucleotidyl transferase kit), 1 × end deoxynucleotide
The reaction system of enzyme reaction buffer solution (offer of terminal deoxynucleotidyl transferase kit) and 1 μM of -1mM dTTP, into system
Microrna and 10U terminal deoxynucleotidyl transferase to be measured, constant temperature 37 DEG C of reactions 30-60min, 85 DEG C of inactivation 25min are added.
4. method according to claim 1, it is characterised in that: it is described for detect Microrna by end deoxynucleotide
In transferase mediated reverse transcription PCR method, reverse transcriptase mediate primer I participate in reverse transcription the following steps are included:
Prepare the upper step reaction product containing 10 times of dilution, 1nM-10 μM of primer I, 1 × reverse transcription buffer (contain Mg2+With
DNTP is provided with Reverse Transcriptase Reagents kit) and reverse transcriptase Mix I (containing reverse transcriptase and RNase inhibitor, with reverse transcription
Enzyme reagent kit provides) reaction system, then 37 DEG C of reaction 15-30min of constant temperature, then 85 DEG C of inactivation 5s.
5. method according to claim 6, it is characterised in that: it is described for detect Microrna by end deoxynucleotide
In transferase mediated reverse transcription PCR method, the design of primer I: primer I includes one section and terminal deoxynucleotidyl transferase
The region for the sequence complementation extended and one section of primer binding zone for amplification.
6. method according to claim 1, it is characterised in that: it is described for detect Microrna by end deoxynucleotide
In transferase mediated reverse transcription PCR method, the PCR amplification step concrete operations for the participation of primer II, III that archaeal dna polymerase mediates
It is as follows:
Reaction carries out in 96PCR pipe, first reverse transcription product of the configuration containing 10 times of dilution, 250nM in the hole in pipe
Plate, is then put by the reaction system of Primer II, 250nM Primer III and 1 × SYBR Premix ExTMTaq IIPCR reaction is carried out in 480Instrument II (Roche) fluorescence quantitative PCR instrument.Program is 95 DEG C of heating
30s, then circulation carries out 95 DEG C of heating 5s, 63 DEG C of extension 30s, terminates reaction after circulation 50 times.
It is described for detecting being mediated by terminal deoxynucleotidyl transferase for Microrna 7. method according to claim 6
In reverse transcription PCR method, it is characterised in that:
The design of PCR amplification primer II, III: the sequence of primer II is identical as target Microrna other than base type
DNA sequence dna；Primer III is consistent with the primer binding zone sequence in primer I for amplification.
8. method according to claim 1, it is described for detect Microrna by terminal deoxynucleotidyl transferase mediate
Detection of the reverse transcription PCR method for miR-122 in hepatic tissue Microrna extracting solution.
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| CN110878335A (en) * | 2019-09-26 | 2020-03-13 | 天津大学 | Method for detecting brain glioma marker based on combination of TdT enzyme and NtbspQI nicking enzyme |
| CN110964763A (en) * | 2019-12-17 | 2020-04-07 | 天津大学 | TDT enzyme-based template-independent RNA3，Method for growing DNA at-OH terminal |
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Application publication date: 20190726 |