WO2018227918A1 - 一种直接定量检测循环miRNA的RT-qPCR方法 - Google Patents

一种直接定量检测循环miRNA的RT-qPCR方法 Download PDF

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WO2018227918A1
WO2018227918A1 PCT/CN2017/117558 CN2017117558W WO2018227918A1 WO 2018227918 A1 WO2018227918 A1 WO 2018227918A1 CN 2017117558 W CN2017117558 W CN 2017117558W WO 2018227918 A1 WO2018227918 A1 WO 2018227918A1
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mirna
poly
reverse transcription
minutes
detection
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苟德明
牛燕琴
康康
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深圳大学
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
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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]

Definitions

  • the invention relates to the field of biomedicine, in particular to an RT-qPCR method for directly quantitatively detecting circulating miRNA without extracting nucleic acid.
  • MicroRNAs are a class of non-coding small RNAs of about 22 nucleotides in length, which are widely found in eukaryotes such as animals, plants, and nematodes.
  • the miRNA regulates gene expression at the post-transcriptional level by binding to the 3' untranslated region (3'-UTR) of the target mRNA, degrading the target mRNA or preventing its translation.
  • 3'-UTR 3' untranslated region
  • miRNAs are widely involved in cell differentiation, proliferation, apoptosis, individual growth and development, and organ formation.
  • the miRNA expression in the organism is finely regulated and has strict space-time specificity. Studies have found that circulating miRNAs are present in the blood and are very stable. More importantly, abnormalities in circulating miRNAs are closely related to the occurrence and development of many diseases, and can be used as novel biomarkers for early diagnosis and prognosis assessment of major diseases such as cancer.
  • RT-qPCR real-time quantitative PCR
  • the more commonly used poly(A) tailing method (Shi R, Biotechnique.2005, 39 (4): 519-525) and Stem loop method (Chen C, Nucleic Acids Res. 2005, 33(20): 1-9).
  • the Poly(A) tailing method uses poly(A) polymerase to bring a poly(A) tail to the 3' end of the miRNA, and then reverse transcription using a primer containing Oligo (dT) sequence. Due to the versatility of the reverse transcription primers, the Poly(A) tailing method reduces the detection cost and also reduces the specificity and sensitivity of the detection.
  • the 5' end of the reverse transcription primer contains a stem-loop structure, and the 3' end usually has 6 specific bases paired with the 3' end of the miRNA, and the reverse transcription reaction can be specifically performed.
  • the stem-loop primer method uses sequence-specific probes, it is relatively expensive in high-throughput miRNA analysis. In addition, depending on the six matching bases, it is obviously insufficient in binding strength, which significantly reduces cDNA synthesis. s efficiency.
  • Kang K et al. invented a new method for detecting miRNA, the S-Poly(T) method, which is respectively referred to in its patent application CN102154505A (referred to as the SS-Oligo (dT) method in the patent, and the primer used is called SS-Oligo ( It is disclosed in dT) Primers and articles (Kang, K, PloS one. 2012.7, e48536.).
  • the primers used are 14-20 base PCR universal primer sequences, 14-20 base universal probe sequences, 8-30 dTs and The specific sequence of the complementary pairing of the 3'-end 3-8 nucleotides of the miRNA molecule of interest.
  • the specificity and sensitivity of the S-Poly(T) method are greatly improved, and the sensitivity is at least 10 times higher.
  • the upgraded version of the S-Poly(T)Plus method (patent application number: 201510558101.5)
  • the miRNA's Poly(A) tailing and reverse transcription one-step reaction is completed, so in terms of ease of operation and reverse transcription efficiency, S-Poly The (T)Plus technology has been further improved and improved, and its overall sensitivity is 2-8 times higher than the S-Poly(T) method.
  • RNA detection methods are based on purified RNA as a template, which will inevitably cause some RNA loss due to incomplete precipitation and recovery of RNA in the extracted nucleic acid.
  • the RNA extraction process is time consuming and prone to contamination and degradation.
  • the present invention comprises the following technical solutions:
  • An RT-qPCR method for directly quantitatively detecting circulating miRNAs which does not need to purify nucleic acids, cleaves miRNAs from protein complexes, and directly performs RT-qPCR detection.
  • RT-qPCR method for directly quantitatively detecting circulating miRNA comprises the following steps:
  • lysis centrifugation The cleavage reagent is used to fully cleave the protein complex in the sample to release the miRNA from the protein complex in the sample; the obtained mixture is centrifuged to obtain a crude RNA, 40 ul The mixture can draw about 35 ul of supernatant;
  • tail-end reverse transcription the crude extract RNA obtained in the step S1 is subjected to Poly(A) tail and S-Poly (T) specific reverse transcription;
  • RT-qPCR quantitative detection RT-qPCR quantitative detection using the reverse transcription product cDNA obtained in step S2 as a template.
  • sample amount in the step S1 is 20 to 50 ul.
  • the reagent for lysis in the step S1 comprises a component: 20 ul 2 ⁇ lysis buffer, 1 ul of proteinase K, and the cleavage reagent correspondingly processes 20 ul of the sample.
  • the 2 ⁇ lysis buffer comprises the following final concentration components: 100 mmol/l Tris-HCl, 300 mmol/l NaCl, 20 mmol/l MgCl 2 ; pH 8.0.
  • the final concentration of the proteinase K was 15 U/mL.
  • lysis conditions were treated at 50 ° C for 20 minutes and then at 95 ° C for 5 minutes.
  • the centrifugation conditions in the step S1 are: 10,000 to 14,000 g, and the mixture is centrifuged at 4 ° C for 5 to 15 minutes; preferably, 13,000 g, and centrifuged at 4 ° C for 5 minutes.
  • reaction system for tailing reverse transcription in the step S2 comprises polyA polymerase and reverse transcriptase.
  • volume percentage of the crude RNA template added in the tailing reverse transcription reaction system in the step S2 is 5 to 75%, preferably 40%.
  • the reaction system for tail-end reverse transcription comprises: 0.5-7.5 uL of supernatant template, 1 ⁇ 0.2 ⁇ L of 0.5 ⁇ mol/L RT primer, 1 ⁇ 0.2 U of PolyA Polymerase, 100 ⁇ 20 U of MMLV, 2.375-0.625 uL reaction buffer, RNase-free Water supplemented to 10 ⁇ L; reaction conditions of tailing reverse transcription: 37 ⁇ 42 ° C for 50 ⁇ 70min, 74 ⁇ 76 ° C for 3 ⁇ 7min to inactivate the enzyme, and then quickly placed on ice , allowed to stand for 2 min to terminate inactivation.
  • the tail-reverse reaction system comprises: 4 uL of supernatant template, 1 ⁇ L of 0.5 ⁇ M RT primer, 1 U of PolyA Polymerase, 100 U of MMLV, 1.5 ⁇ L of reaction buffer, RNase-free Water to 10 ⁇ L;
  • the reaction conditions for reverse transcription were: incubation at 37 ° C for 30 min, incubation at 42 ° C for 30 min, incubation at 75 ° C for 5 min to inactivate the enzyme, then quickly placed on ice and allowed to stand for 2 min to terminate inactivation.
  • the real-time PCR is quantitatively detected by using cDNA as a template, and the DNA polymerase used in the process is a hot start enzyme to reduce non-specific amplification;
  • the real-time PCR reaction system is: 4 ⁇ qPCR reaction Buffer: 5 ⁇ L, 1 ⁇ mol/L Forward Primer 4 ⁇ L, 10 ⁇ mol/L universal reverse primer 0.4 ⁇ L, 10 ⁇ mol/L universal Taqman probe 0.5 ⁇ L, 100 ⁇ ROX Rerference Dye 0.2 ⁇ L, hotstart Alpha Taq Polymerase 0.0125 ⁇ L, cDNA 0.5 ⁇ L, RNase-free Water was added to 20 ⁇ L; the reaction conditions were: pre-denaturation at 95 ° C for 5 minutes, denaturation at 95 ° C for 10 s, annealing at 60 ° C for 40 s, 40 cycles.
  • the hot start enzyme is prepared by mixing the DNA polymerase and the hot start antibody in an equal volume and allowing to stand at room temperature for 6 hours.
  • the sample includes serum, plasma/serum, urine, tears, milk, saliva, sputum or stool extract supernatant; preferably the sample is plasma.
  • the step of extracting nucleic acid is not required, and the miRNA is quantitatively detected, and the flow chart thereof is shown in FIG. 1 .
  • the operation is simple, the time is shortened, the time for preparing cDNA is reduced by at least 70%, and the simplicity is superior to the conventional method.
  • the Direct S-Poly(T)Plus method of the present invention has a wider range of template requirements in the reverse transcription step, and 5%-75% of the crude extract RNA can satisfy the reverse transcription requirement, and the transcription efficiency is superior to the conventional method.
  • the technical system of the present invention is particularly suitable for detecting miRNAs from biological fluid samples having low miRNA abundance.
  • the sensitivity of the method of the invention is significantly higher than conventional methods. For example, in terms of sensitivity, 20 ul of body fluid samples can detect 175 miRNAs.
  • the Direct S-Poly(T)Plus method of the present invention can efficiently detect miRNAs from biological fluid samples including serum, plasma/serum, urine, milk, saliva, sputum, stool extract supernatant, and cell culture fluid. Detection efficiency is an order of magnitude higher than traditional methods, thereby increasing the sensitivity and accuracy of quantitative detection of humoral miRNAs.
  • the simplicity, sensitivity and specificity of the present invention have important application prospects in early disease screening and prognosis evaluation, and can be widely used for early non-invasive screening of tumors, cardiovascular diseases or other major diseases.
  • Figure 1 shows the direct quantitative RT-qPCR fluorescence detection process (Direct S-Poly(T)Plus). Among them, in the tail-end reverse transcription system, 4 ul crude RNA was used as a template as the optimal solution.
  • Figure 2 shows the effect of different cracking schemes in the Direct S-Poly(T)Plus method.
  • Figure 3 shows the difference between the one-step method (one-step completion of the tailing and reverse transcription reactions) and the two-step method (the reverse transcription reaction after the tail is added) in the Direct S-Poly(T)Plus method.
  • Figure 4 shows the initial crude RNA addition ratio in the Direct S-Poly(T)Plus method.
  • Figure 5 compares the amount of miRNA expression in serum plasma of the same volunteer with Direct S-Poly(T)Plus. ***P ⁇ 0.001.
  • Figure 6 uses the extracted RNA as a template to compare the expression levels of miRNA in serum and plasma of the same volunteers using the S-Poly(T)Plus method. Normalized internal reference was made with miR-cel-54, ***P ⁇ 0.001.
  • Figure 7 shows the effect of hot-starting Alpha Taq Polymerase on non-specific amplification in the Direct S-Poly(T)Plus method.
  • Figure 8 is a hsa-miR-15b-5p amplification curve, -RT: a negative control without reverse transcriptase, and the detection method is Direct S-Poly(T)Plus.
  • Figure 9 shows the effect of the amount of hot-start Alpha taq polymerase on the Ct value of miRNA detection (20 ul system).
  • the detection method is Direct S-Poly (T) Plus.
  • Figure 10 is a negative control (no reverse transcriptase) amplification curve (20 ul system) using 0.4 ul Hotstart Alpha Taq Polymerase miRNA as Direct S-Poly (T) Plus.
  • Figure 11 is a negative control (no reverse transcriptase) amplification curve (20 ul system) using 0.0125 ul of Hotstart Alpha taq Polymerase miRNA as Direct S-Poly (T) Plus.
  • Figure 12 shows the sensitivity and linear range of the Direct S-Poly(T)Plus method.
  • Figure 13 shows the sensitivity comparison of three miRNA detection methods.
  • Figure 14 is a single sample validation of miRNAs that showed significant changes in primary screening for colorectal cancer.
  • the verification method is Direct S-Poly(T)Plus.
  • the data is ⁇ SE, **P ⁇ 0.01, ***P ⁇ 0.001, ns, not significant.
  • Figure 15 is a single sample validation of miRNAs that showed significant changes in primary screening for colorectal cancer.
  • the verification method is S-Poly(T)Plus (internal reference is miR-cel-54).
  • the data is ⁇ SE, **P ⁇ 0.01, ***P ⁇ 0.001, ns, not significant.
  • the blood comes from Shenzhen People's Hospital and Peking University Shenzhen Hospital.
  • the plasma collection procedure was as follows: blood collection was performed on a blood collection tube containing EDTA anticoagulant, and centrifuged at 3,000 rpm for 10 minutes at 4 ° C, and the supernatant was plasma; the whole blood sample was allowed to stand at room temperature for 1 hour, serum was taken at 4 ° C, and centrifuged at 3,000 rpm for 10 minutes. The supernatant is serum. Serum/plasma samples were dispensed in a 20-50 ul system and stored at -80 °C.
  • the optimal scheme of miRNA direct RT-qPCR fluorescence quantitative detection method (Direct S-Poly (T) Plus) is shown in Figure 1.
  • 20 ul of plasma can prepare 35 ul of crude RNA, corresponding to 87.5 ul of cDNA.
  • an average of 175 miRNAs can be detected. Ignoring the operating time, the entire miRNA detection process takes only 140 minutes.
  • Example 1 Direct S-Poly(T)Plus method for comparison of circulating miRNA content in plasma and serum
  • the same volunteer serum and plasma were simultaneously used as templates, and 10 pairs of serum and plasma samples of the same healthy volunteer were collected.
  • the amount of miRNA expression in an equal amount of serum or plasma samples was separately measured by the Direct S-Poly(T)Plus method of the present invention. Specifically, it includes the following steps:
  • tail-end reverse transcription miRNA plus Poly (A) tail and reverse transcription (synthesis of first-strand cDNA) were carried out in one reaction system, and S-Poly (T) primers were used for reverse transcription of miRNA.
  • the 2 ⁇ lysis buffer contains the following final concentrations of components: 100mmol / lTris-HCl, 300mmol / l NaCl, 20mmol / l MgCl 2; pH 8.0; final concentration of the proteinase K was 15U / mL.
  • the tail-reverse transcription reaction system consists of: 4uL of crude RNA, 1 ⁇ L of 0.05 ⁇ M RT primer (reverse transcription primer), 1U of PolyA Polymerase (polyadenylation polymerase), 100U of MMLV (murine leukemia reverse transcriptase) 1.5 ⁇ L of reaction buffer (reaction buffer) and RNase-free Water (without RNase water) were added to 10 ⁇ L.
  • the reaction buffer contained the following final concentration components: 200 mM Tris-HCl, 600 mM NaCl, 40 mM MgCl 2 , 4 mM ATP, 2 mM dNTP, pH 8.0.
  • reaction conditions of tailing reverse transcription were as follows: incubation at 37 ° C for 30 min, incubation at 42 ° C for 30 min, incubation at 75 ° C for 5 min to inactivate the enzyme, and then quickly placed on ice, allowed to stand for 2 min to terminate inactivation.
  • the S-Poly(T) primer consists of four parts, and the sequence from the 5' end to the 3' end is: a 14-20 base PCR universal primer sequence, and a 14-20 base universal probe sequence. , 11 oligo (dT) and 5-7 specific bases paired with miRNA 3'. More preferably, the S-Poly(T) primer sequence is from the 5' end to the 3' end: 16 base PCR universal primer sequence, 17 base universal probe sequence, 11 oligo (dT) And 6 specific bases paired with miRNA 3'.
  • the sequence of the miRNA detected in the present invention is derived from miRBase, and different S-Poly(T) primers and upstream primers are designed according to the respective sequences, and the S-Poly(T) primer sequences for detecting different miRNAs are shown in Table 1.
  • the miRNA-specific upstream primer is a miRNA-specific sequence that does not contain a 3'-end 3-8 base
  • the downstream universal primer of the miRNA is derived from a 14-20 base universal primer sequence of the S-Poly(T) primer.
  • Real-time PCR quantitative detection uses probe method or SYBR fluorescent dye method.
  • the probe method was employed, and the probe used was a universal probe whose sequence was derived from a 14-20 base PCR universal primer sequence on the S-Poly(T) primer.
  • the reaction system of Real-time PCR is as follows:
  • the PCR operation instrument was ABI StepOnePlus thermal cycler, and the reaction conditions were: pre-denaturation at 95 ° C for 5 minutes, denaturation at 95 ° C for 10 s, annealing at 60 ° C for 40 s, and 40 cycles. Two replicate wells per PCR reaction. Data analysis was performed using GraphPad Prism 5 software and the test method was two-tailed Student's test. The final result is expressed as mean ⁇ SD (standard deviation).
  • the S-Poly(T)Plus method for detecting circulating miRNAs requires the extraction of nucleic acids, including the following steps:
  • serum/plasma total RNA is extracted, and the specific steps are as follows:
  • 0.1 pM nematode miRNA cel-miR-54 was added as an internal reference to 1 mL of RNAiso-Plus (TaKaRa) in advance, 100 ⁇ L of serum/plasma was added, mixed by pipetting, and allowed to stand at room temperature for 5 minutes; 200 ⁇ L of chloroform was added to cover the centrifuge tube cap. , shaking vigorously for 20 seconds; standing at room temperature for 5 minutes;
  • the precipitate is dried at room temperature for 2 to 3 minutes, dissolved in 100 ⁇ L of RNase-free Water, and the lysate is stored at -80 ° C, or directly subjected to real-time PCR detection of miRNA.
  • the S-Poly(T)Plus method detects miRNA, using reverse transcription primers and qPCR primers as in Table 1 of Example 1, including the following steps:
  • tail-end reverse transcription miRNA plus Poly (A) tail and reverse transcription (synthesis of first-strand cDNA) were carried out in one reaction system, and S-Poly (T) primers were used for reverse transcription of miRNA.
  • the reaction system with tailing reverse transcription contains: 4 ⁇ L of serum total RNA, 1 ⁇ L of 0.05 ⁇ M RT primer (reverse transcription primer), 1 U of PolyA Polymerase (polyadenylation polymerase), 100 U of MMLV (murine leukemia reverse transcriptase) 2.5 ⁇ L of reaction buffer (reaction buffer) and RNase-free Water (without RNase water) were added to 10 ⁇ L.
  • the reaction buffer contained 200 mM Tris-HCl, 600 mM NaCl, 40 mM MgCl2, 4 mM ATP, 2 mM dNTP, pH 8.0.
  • reaction conditions of tailing reverse transcription were as follows: incubation at 37 ° C for 30 min, incubation at 42 ° C for 30 min, incubation at 75 ° C for 5 min to inactivate the enzyme, and then quickly placed on ice, allowed to stand for 2 min to terminate inactivation.
  • the reaction system of Real-time PCR is as follows:
  • the PCR operation instrument was ABI StepOnePlus thermal cycler, and the reaction conditions were: pre-denaturation at 95 ° C for 3 minutes, denaturation at 95 ° C for 10 s, annealing at 60 ° C for 30 s, and 40 cycles. Two replicate wells per PCR reaction. The relative expression amount in this example was calculated using 2- ⁇ Ct. Data analysis was performed using GraphPad Prism 5 software and the test method was two-tailed Student's test. The final result is expressed as mean ⁇ SD (standard deviation).
  • miRNAs can be cleaved from the protein complex by any of the following six treatments:
  • lysis system 20ul lysate, 20ul sample; lysis conditions: 75 ° C for 5 minutes;
  • lysis system 20ul lysate, 1ul proteinase K, 20ul sample; lysis conditions: 50 ° C treatment for 20 minutes, then 95 ° C for 5 minutes;
  • lysis system 20ul 2 ⁇ lysis buffer, 1ul proteinase K, 20ul sample; lysis conditions: 50 ° C treatment for 20 minutes, then 95 ° C for 5 minutes;
  • lysis system 10 ul 2 ⁇ lysis buffer, 10 ul lysate, 1 ul of proteinase K, 20 ul sample; lysis conditions: 50 ° C treatment for 20 minutes, and then 95 ° C for 5 minutes.
  • the lysate described in the above treatments included the following final concentration components: 2.5% tween-20, 50 mM Tris and 1 mM EDTA; the 2X lysis buffer contained the following final concentration of components: 100 mmol/l Tris-HCl, 300 mmol/l NaCl, 20 mmol/l MgCl 2 ; pH 8.0; the final concentration of the proteinase K was 15 U/mL.
  • the one-step sensitivity is greatly improved compared to the two-step method.
  • the two-step method, miRNA Poly (A) is followed by reverse transcription; the one-step method, miRNA Poly (A) tailing and reverse transcription, is carried out in the same reaction.
  • the crude RNA was used as a template, and the same procedure as in Example 1 was carried out, and the sensitivity of the two-step method and the one-step method was again compared.
  • the sensitivity of the one-step method is improved by 2.5 to 52 times (1.7 to 5.7 Ct value difference) compared with the two-step method (Fig. 3). .
  • Crude RNA may contain some components that inhibit Poly(A) tailing and reverse transcriptase activity, so the initial amount of RNA extracted in the Direct S-Poly(T)Plus method has an effect on the sensitivity of the method.
  • Different crude extraction RNA starting amounts the test operation is the same as in Example 1, and the test results are shown in Fig. 4. It can be seen that when the initial volume percentage of crude extraction RNA increases from 0.5% to 40%, the Ct value of miRNA is linear. reduce. However, when the proportion of crude RNA addition increased to 60% and 75%, the Ct value of miRNA increased. In the present invention, 40% of the crude RNA starting amount is recommended as the optimum ratio.
  • RNA purification may introduce some genomic DNA contamination, so in qPCR, mismatch with genomic DNA is more likely to occur.
  • One effective way to reduce non-specific amplification is hot start, which prevents or reduces DNA synthesis before the onset of thermal cycling.
  • hot start This example compares the common DNA polymerase and the hot-start DNA polymerase used in the PCR portion of the Direct S-Poly(T)Plus method.
  • an effective method of forming a hot start that is, a Taq enzyme antibody, which binds to the DNA polymerase, is not activated before the start of the thermal cycle.
  • the hot-start DNA polymerase used in this embodiment is Hotstart Alpha Taq Polymerase, and the specific preparation method is Alpha Taq Polymerase (VitaNavi, St. Louis USA) and Taq Antibody (Fei Peng Company, Shenzhen) is mixed in an equal volume, and left at room temperature for 6 hours. .
  • the use of a hot start enzyme can effectively reduce non-specific amplification.
  • the direct amplification efficiency of miRNA was analyzed for the amount of enzyme of Hotstart Alpha Taq Polymerase.
  • the activity of Hotstart Alpha Taq Polymerase is very high, and the amount of 0.0125 uL of enzyme in the 20 ul PCR system can meet the amplification requirements.
  • Example 7 Effect of the amount of Hotstart Alpha Taq Polymerase on the non-specific amplification occurring in the Direct S-Poly(T)Plus method
  • Example 8 Direct S-Poly(T)Plus method for detecting linear gradient range of plasma miRNA
  • This example analyzes the linear gradient range of plasma miRNA detected by the Direct S-Poly(T)Plus method. Serum RNA was subjected to 4-fold serial dilution (the amount of initial plasma corresponding to the total RNA usage was 0.1-0.0004 ul), and then detected.
  • the Direct S-Poly(T)Plus method detects plasma miRNAs (hsa-miR-451a, hsa-miR-21-5p, hsa-miR-126-3p, hsa-miR-92a-3p, hsa Both -miR-210-3p, hsa-miR-27b-3p, hsa-miR-103a-3p and hsa-miR-92a-3p) have a good linear correlation coefficient R2 (0.9139-0.9988). Therefore, the Direct S-Poly(T)Plus method detects plasma miRNAs with a good linear relationship and a wide dynamic range.
  • the Direct S-Poly(T)Plus method will be compared with the most popular Stem-loop method and the S-Poly(T)Plus method in Comparative Example 1.
  • the Stem-loop and S-Poly(T)Plus methods use purified RNA as a template.
  • the S-Poly(T)Plus method was the same as in Example 1, and the Stem-loop method was followed according to the kit TaqMan microRNA assay kit (Applied Biosystems) instructions.
  • three miRNA detection methods are used to detect six miRNAs, namely hsa-miR-140-5p, hsa-miR-124a-3p, hsa-miR-16-5p, hsa-miR-93-5p, hsa -miR-25-3p and hsa-miR-106-5p.
  • the Ct values of hsa-miR-16-5p (25.43) and hsa-miR-93-5p (27.78) were slightly smaller in the S-Poly(T)Plus method, and the remaining miRNA Ct values were The smallest in the Direct S-Poly(T)Plus method.
  • the Direct S-Poly(T)Plus method is 7-342 times more sensitive (2.8-8.4 Ct values) than the stem-loop method.
  • the present invention introduces a sensitive miRNA detection method that does not require RNA extraction, that is, direct fluorescent quantitative PCR amplification technology of miRNA (Direct S-Poly(T)Plus, referred to as DSPP for short. ).
  • Direct S-Poly(T)Plus the miRNA is first released from the protein complex to obtain the crude RNA; then, based on the S-Poly(T)Plus method, the crude RNA is simultaneously added to the same reaction system. Tail and reverse transcription. Ignoring the operation time, the cDNA can be prepared in 95 minutes by the Direct S-Poly(T)Plus method of the present invention, and the entire miRNA detection process can be completed in 140 minutes by adding qPCR time.

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Abstract

一种不需提取核酸，直接检测血清或血浆中循环miRNA的实时荧光定量RT-qPCR方法。所述方法包括：S1：裂解血清或血浆中的外泌体及miRNA蛋白复合体，离心后得到循环miRNA粗提物；S2：miRNA加尾及逆转录；S3：RT-qPCR定量检测。该方法不需要提取核酸，且miRNA的Poly(A)加尾和逆转录将在一个反应体系中同步完成，操作简便，缩短时间，可在95分钟内完成cDNA的制备。该方法灵敏度与stem-loop方法相比提高数十甚至上百倍，建立了一种非常简便、灵敏、高效、快捷、廉价的miRNA检测的技术体系，尤其适合于临床应用推广及其从miRNA丰度较低的生物体液样本中检测miRNA。

Description

一种直接定量检测循环miRNA的RT-qPCR方法 技术领域
本发明涉及生物医学领域，具体涉及一种不需提取核酸，直接定量检测循环miRNA的RT-qPCR方法。
背景技术
MicroRNA(miRNA)是一类长约22个核苷酸非编码小RNA，广泛存在于动物、植物、线虫等真核生物中。miRNA通过与靶mRNA的3’端非翻译区(3’-UTR)结合，降解靶mRNA或者阻止其翻译，从而在转录后水平上调控基因的表达。功能上，miRNA广泛参与细胞的分化、增殖、凋亡、个体生长发育以及器官形成。生物体内miRNA表达被精细调控，具有严格的时空特异性。研究发现，血液中存在着循环miRNA且非常稳定，更为重要是，循环miRNA的异常与许多疾病的发生发展密切相关，可作为癌症等重大疾病早期诊断和预后评估的新型生物标记物。
长期以来，基于实时荧光定量PCR(RT-qPCR)的检测技术一直都被认为是最灵敏的miRNA检测手段之一，比较常用的有poly(A)加尾法(Shi R,Biotechnique.2005,39(4):519-525)和茎环引物(Stem loop)法(Chen C,Nucleic Acids Res.2005,33(20):1-9)。Poly(A)加尾法是利用poly(A)聚合酶使miRNA的3’端带上一段poly(A)尾巴，然后用含有Oligo(dT)序列引物进行逆转录。由于逆转录引物的通用性，因此Poly(A)加尾法在降低检测成本的同时，也降低了检测的特异性和灵敏性。茎环引物法中逆转录引物5’端含有 一个茎环结构，3’端通常具有6个与miRNA3’端配对的特异性碱基，可以特异性的进行逆转录反应。但是由于茎环引物法使用的是序列特异性探针，在高通量的miRNA分析中成本相对昂贵，此外，依赖于6个匹配碱基在结合力度方面显然是不够的，会显著降低cDNA合成的效率。
Kang K等发明了一种新型的检测miRNA的方法S-Poly(T)法，分别在其专利申请CN102154505A(在该专利中称为S-S-Oligo(dT)法，所用引物称为S-S-Oligo(dT)引物)和文章(Kang,K,PloS one.2012.7,e48536.)中进行了公开。在S-Poly(T)方法中，所用引物从5’端开始依次是14-20个碱基的PCR通用引物序列，14-20个碱基的通用探针序列，8-30个dT及与目的miRNA分子的3’端3-8个核苷酸互补配对的特异性序列。与poly(A)加尾法和茎环引物法相比，S-Poly(T)方法的特异性和灵敏度都大大提高，灵敏度至少提高10倍以上。在升级版的S-Poly(T)Plus方法中(专利申请号：201510558101.5)，miRNA的Poly(A)加尾和逆转录一步反应完成，因此在操作简便性和逆转录效率方面，S-Poly(T)Plus技术又有进一步改进和提高，其总体灵敏度比S-Poly(T)方法提高2-8倍。
现有miRNA检测方法都是基于纯化的RNA为模板，由于提取核酸中RNA沉淀和回收的不完全，将会不可避免的造成一些RNA丢失。此外，RNA提取过程费时，易造成污染和降解。
可见，现有技术还有待完善。
发明内容
鉴于此，有必要针对上述问题提供一种不需提取核酸，更为简便、灵敏、高效、廉价的直接定量检测循环miRNA的RT-qPCR方法，称之为Direct S-Poly(T)Plus(DSPP)。
为了实现上述发明目的，本发明包含以下技术方案：
一种直接定量检测循环miRNA的RT-qPCR方法，所述方法不需提纯核酸，将miRNA从蛋白复合体中裂解出来，直接进行RT-qPCR检测。
进一步的，所述直接定量检测循环miRNA的RT-qPCR方法，包含以下步骤：
S1、裂解离心：利用裂解用试剂将样本中的蛋白复合体中充分裂解，使miRNA从样本中的蛋白复合体中释放出来；将所得的混合物离心，得上清即为粗提RNA，40ul的混合物可以吸取约35ul上清；
S2、加尾逆转录：将所述步骤S1中所获得粗提RNA进行加Poly(A)尾及S-Poly(T)特异性逆转录；
S3、RT-qPCR定量检测：以步骤S2中获得的逆转录产物cDNA为模板进行RT-qPCR定量检测。
进一步的，所述步骤S1中所述样本用量为20～50ul。
进一步的，所述步骤S1中裂解用试剂包括组分：20ul 2×lysis buffer、1ul蛋白酶K，所述裂解用试剂对应处理20ul样本。
进一步的，所述2×lysis buffer包含以下终浓度的组分：100mmol/lTris-HCl、300mmol/l NaCl、20mmol/l MgCl 2；pH为8.0。
进一步的，所述蛋白酶K的终浓度为15U/mL。
进一步的，所述裂解条件为50℃处理20分钟，然后95℃保持5 分钟。
进一步的，所述步骤S1中的离心条件为：10，000～14，000g，4℃条件下离心5～15分钟；优选13,000g，4℃条件下离心5分钟。
进一步的，所述步骤S2中加尾逆转录的反应体系包含多聚腺苷酸聚合酶(polyA polymerase)和逆转录酶(reverse transcriptase)。
进一步的，所述步骤S2中加尾逆转录反应体系中所加入粗提RNA模板的体积百分比为5～75％，优选40％。
进一步优选地，加尾逆转录的反应体系包含：0.5-7.5uL上清模板、1±0.2μL的0.5μmol/L RT primer、1±0.2U的PolyA Polymerase、100±20U的MMLV、2.375-0.625uL的reaction buffer、RNase-free Water补足至10μL；加尾逆转录的反应条件为：37～42℃保温50～70min，74～76℃保温3～7min以灭活酶，然后迅速置于冰上，静置2min以终止灭活。
进一步优选地，加尾逆转录的反应体系包含：4uL上清模板，1μL的0.5μM RT primer，1U的PolyA Polymerase，100U的MMLV，1.5μL的reaction buffer，RNase-free Water补足至10μL；加尾逆转录的反应条件为：37℃保温30min，42℃保温30min，75℃保温5min以灭活酶，然后迅速置于冰上，静置2min以终止灭活。
进一步的，所述步骤S3中以cDNA为模板进行real-time PCR定量检测，此过程使用的DNA聚合酶为热启动酶，目的是减少非特异性扩增；real-time PCR反应体系为：4×qPCR reaction Buffer：5μL、1μmol/L Forward Primer 4μL、10μmol/L universal reverse  primer0.4μL、10μmol/L universal Taqman probe0.5μL、100×ROX Rerference Dye0.2μL、hotstart Alpha Taq Polymerase0.0125μL、cDNA0.5μL、RNase-free Water加至20μL；反应条件为：预变性95℃5分钟，变性95℃10s，退火60℃40s，40个循环。
进一步的，所述热启动酶的制备方法为：将DNA聚合酶和热启动抗体等体积混合，室温放置6小时。
进一步的，所述样本包括血清、血浆/血清、尿液、眼泪、乳汁、唾液、痰液或粪便抽提上清；优选样本为血浆。
本发明有益效果：
1、本发明Direct S-Poly(T)Plus方法中，不需要提取核酸的步骤，定量检测miRNA，其流程图如图1所示。操作简便，缩短时间，制备cDNA的时间至少减少70％以上，简便性优于传统方法。
2、本发明Direct S-Poly(T)Plus方法在逆转录这一步对于模板量要求范围更宽，5％-75％的粗提RNA均可满足逆转录要求，转录效率优于传统方法。
3、本发明中的技术体系尤其适合于从miRNA丰度较低的生物体液样本中检测miRNA。本发明方法的灵敏性显著高于传统方法。比如灵敏性方面，20ul的体液样本就可以实现175个miRNA的检测。
4、本发明Direct S-Poly(T)Plus方法能从包括血清、血浆/血清、尿液、乳汁、唾液、痰液、粪便抽提上清及细胞培养液等生物体液样本中高效检测miRNA，检测效率比传统方法高出一个数量级，从而提高了体液miRNA定量检测的灵敏性和准确性。
5、本发明的简便性、灵敏性和特异性使其在疾病早期筛查和预后评估等研究方面有重要应用前景，可广泛用于肿瘤、心血管病或其它重大疾病的早期无创筛查。
附图说明
图1为miRNA直接RT-qPCR荧光定量检测流程(Direct S-Poly(T)Plus)。其中，在加尾逆转录体系中，4ul粗提RNA作为模板为最优方案。
图2为Direct S-Poly(T)Plus方法中不同裂解方案的效果比较。
图3为Direct S-Poly(T)Plus方法中一步法(加尾和逆转录反应一步完成)和两步法(先加尾后进行逆转录反应)的差别。
图4为Direct S-Poly(T)Plus方法中起始粗提RNA加入比例。
图5用Direct S-Poly(T)Plus比较同一志愿者的血清血浆中miRNA表达量。***P<0.001。
图6以提取的RNA为模板，用S-Poly(T)Plus方法比较同一志愿者血清和血浆中miRNA的表达量。用miR-cel-54做归一化内参，***P<0.001。
图7为热启动Alpha Taq Polymerase对Direct S-Poly(T)Plus方法中非特异性扩增减少作用。
图8为hsa-miR-15b-5p扩增曲线,-RT:不加逆转录酶的阴性对照，检测方法为Direct S-Poly(T)Plus。
图9为热启动Alpha taq polymerase用量对miRNA检测Ct值的影响(20ul体系)，检测方法为Direct S-Poly(T)Plus。
图10为使用0.4ul Hotstart Alpha Taq Polymerase miRNA的阴性对照(不加逆转录酶)扩增曲线(20ul体系)，检测方法为Direct S-Poly(T)Plus。
图11为使用0.0125ul Hotstart Alpha taq Polymerase miRNA的阴性对照(不加逆转录酶)扩增曲线(20ul体系)，检测方法为Direct S-Poly(T)Plus。
图12为Direct S-Poly(T)Plus方法的灵敏度和线性范围。
图13为三种miRNA检测方法灵敏度比较。
图14为六个在结直肠癌初筛中显著变化的miRNA单个样本验证。验证方法为Direct S-Poly(T)Plus。数据为±SE,**P<0.01,***P<0.001,ns,不显著。
图15为六个在结直肠癌初筛中显著变化的miRNA单个样本验证。验证方法为S-Poly(T)Plus(内参为miR-cel-54)。数据为±SE,**P<0.01,***P<0.001,ns,不显著。
具体实施方式
为了更好地说明本发明所解决的问题、所采用的技术方案和所达到的效果，现结合具体实施例和相关资料进一步阐述。需要说明的是，本发明内容包含但不限于以下实施例及其组合实施方式。
如无特别说明，以下实施例中所采用的各种原料均来源于市场销售，所采用的方法均为常规方法，其中引物、探针来自美国Integrated DNA Technologies(IDT)公司。
本申请中主要材料来源如下：
血液来源于深圳市人民医院和北京大学深圳医院。血浆收集流程为：采血于含有EDTA抗凝剂的采血管，4℃，3,000转离心10分钟，上清即为血浆；全血样本室温放置1小时，取血清4℃，3,000转离心10分钟，上清即为血清。血清/血浆样本分装为20-50ul的体系，保存于-80℃。
miRNA直接RT-qPCR荧光定量检测方法(Direct S-Poly(T)Plus)最优方案流程如图1。在最优的方案中，20ul的血浆可以制备35ul粗提RNA，对应87.5ul cDNA。按照20ul qPCR体系加入0.5ulcDNA，平均可以检测175个miRNA。忽略操作时间，整个miRNA检测流程只需140分钟。
实施例1 Direct S-Poly(T)Plus方法比较血浆和血清中的循环miRNA含量
在本实施例中，同一志愿者血清和血浆同时作为模板，共收集了同一健康志愿者的血清和血浆样本10对。用本发明中Direct S-Poly(T)Plus方法分别检测等量血清或者血浆样本中的miRNA表达量。具体包含以下步骤：
S1、裂解离心，具体步骤为：
1)20uL血浆/血清与20uL 2×lysis buffer混合均匀，加入1uL蛋白酶K，50℃处理20分钟，然后95℃保持5分钟，置于冰上；
2)13,000g，4℃离心5分钟；吸取上清液(粗提RNA)转移至另一新的离心管中或直接用于S2；
S2、加尾逆转录：miRNA加Poly(A)尾和逆转录(第一链cDNA 的合成)在一个反应体系中进行，利用S-Poly(T)引物进行miRNA的逆转录。
所述2×lysis buffer包含以下终浓度的组分：100mmol/lTris-HCl、300mmol/l NaCl、20mmol/l MgCl 2；pH为8.0；所述蛋白酶K的终浓度为15U/mL。
加尾逆转录的反应体系包含：4uL粗提RNA，1μL的0.05μM RT primer(逆转录引物)，1U的PolyA Polymerase(多聚腺苷酸聚合酶)，100U的MMLV(鼠白血病逆转录酶)，1.5μL的reaction buffer(反应缓冲液)，RNase-free Water(无RNA酶水)补足至10μL。所述reaction buffer包含以下终浓度的组分：200mM Tris-HCl，600mM NaCl，40mM MgCl 2，4mM ATP，2mM dNTP，pH 8.0。加尾逆转录的反应条件为：37℃保温30min，42℃保温30min，75℃保温5min以灭活酶，然后迅速置于冰上，静置2min以终止灭活。
所述S-Poly(T)引物由四部分组成，其序列从5’端到3’端依次为：14-20个碱基的PCR通用引物序列、14-20个碱基的通用探针序列、11个oligo(dT)和5-7个与miRNA 3’配对的特异性碱基。更优选地，所述S-Poly(T)引物序列从5’端到3’端依次为：16个碱基的PCR通用引物序列、17个碱基的通用探针序列、11个oligo(dT)和6个与miRNA 3’配对的特异性碱基。
本发明中所检测的miRNA的序列来自于miRBase，根据各自序列设计不同的S-Poly(T)引物、上游引物，检测不同miRNA的S-Poly(T)引物序列如表1所示。
表1、本发明中所使用的引物和探针
Figure PCTCN2017117558-appb-000001
Figure PCTCN2017117558-appb-000002
S3、PCR：以步骤S2中逆转录获得的第一链cDNA为模板，用miRNA特异上游引物和下游通用引物进行real-time PCR定量检测。所述miRNA特异上游引物是不含3’端3-8个碱基的miRNA特异序列，所述miRNA的下游通用引物来自于S-Poly(T)引物的14-20个碱基的通用引物序列。
Real-time PCR定量检测采用探针法或者SYBR荧光染料法。本实施例中采用探针法，所用探针为通用探针，其序列来自于S-Poly(T)引物上14-20个碱基的PCR通用引物序列。Real-time PCR的反应体系如下：
组分 含量
4×qPCR reaction Buffer(Geneup，μL) 5
1μM Forward Primer(μL) 4
10μM universal reverse primer(μL) 0.4
10μM universal Taqman probe(μL) 0.5
100×ROX Rerference Dye(μL) 0.2
hotstart Alpha Taq Polymerase(Geneup，μL) 0.0125
cDNA(μL) 0.5
RNase-free Water up to(μL) 20
PCR运行仪器为ABI StepOnePlus thermal cycler，反应条件为： 预变性95℃5分钟，变性95℃10s，退火60℃40s，40个循环。每个PCR反应两个复孔。数据分析使用GraphPad Prism 5软件，检验方法为two-tailed Student's test。最终结果用平均值±SD(标准差)表示。
结果表明，血清和血浆样本都可用于miRNA直接定量RT-qPCR检测，但是血浆中miRNA检测Ct值全部显著低于血清，说明miRNA在血浆中的表达量显著高于血清(图5)。
对比例1、S-Poly(T)Plus方法检测循环miRNA
S-Poly(T)Plus法检测循环miRNA需要提取核酸，包括以下步骤：
(一)、提取血清/血浆总RNA
在本实施例中提取血清/血浆总RNA，具体步骤为：
1)0.1pM线虫miRNA cel-miR-54作为内参提前加入1mL的RNAiso-Plus(TaKaRa)中，加入100μL血清/血浆，吹打混匀，室温静置5分钟；加入200μL氯仿，盖紧离心管盖，剧烈振荡20秒；室温静置5分钟；
2)12,000g，4℃离心15分钟；小心取出离心管，此时匀浆液分为三层，即：无色的上清液(含miRNA)、中间的白色蛋白层、及有颜色的下层有机相；吸取500μL上清液转移至另一新的1.5mL离心管中；
3)向上清液中加入5μL适当浓度的糖原(Applichem)溶液，使糖原终浓度为15μg/mL，再加入与等体积的异丙醇(505μL)，上下颠倒充分混匀，-20℃或-80℃静置至少10分钟；
4)13,500g，4℃离心10分钟；弃去上清液，向沉淀中加入1mL的75％乙醇，轻轻颠倒清洗沉淀；13,500g，4℃离心5分钟，完全弃去上清，如管壁上沾有残余溶液，应再次离心并弃尽上清；
5)沉淀室温干燥2～3分钟，加入100μL RNase-free Water溶解，溶解产物置于-80℃储存，或者直接进行miRNA的荧光定量PCR检测。
(二)、S-Poly(T)Plus法检测miRNA
S-Poly(T)Plus法检测miRNA，使用逆转录引物和qPCR引物同实施例1表1，包括以下步骤：
S1、加尾逆转录：miRNA加Poly(A)尾和逆转录(第一链cDNA的合成)在一个反应体系中进行，利用S-Poly(T)引物进行miRNA的逆转录。
加尾逆转录的反应体系包含：4μL血清总RNA，1μL的0.05μM RT primer(逆转录引物)，1U的PolyA Polymerase(多聚腺苷酸聚合酶)，100U的MMLV(鼠白血病逆转录酶)，2.5μL的reaction buffer(反应缓冲液)，RNase-free Water(无RNA酶水)补足至10μL。所述reaction buffer包含200mM Tris-HCl，600mM NaCl，40mM MgCl2，4mM ATP，2mM dNTP，pH 8.0。加尾逆转录的反应条件为：37℃保温30min，42℃保温30min，75℃保温5min以灭活酶，然后迅速置于冰上，静置2min以终止灭活。
S2、以步骤S1中逆转录获得的第一链cDNA为模板，Real-time PCR定量检测采用探针法，所用探针为通用探针，其序列同实施例1。 Real-time PCR的反应体系如下：
组分 含量
4×qPCR Reaction Buffer(Geneup，μL) 5
1μM Forward Primer(μL) 4
10μM universal reverse primer(μL) 0.4
10μM universal Taqman probe(μL) 0.5
100×ROX Rerference Dye(μL) 0.2
Hotstart SM Taq Polymerase(Geneup，U) 0.5
Diluted cDNA(μL) 0.5
RNase-free Water up to(μL) 20
PCR运行仪器为ABI StepOnePlus thermal cycler，反应条件为：预变性95℃3分钟，变性95℃10s，退火60℃30s，40个循环。每个PCR反应两个复孔。本实施例中相对表达量用2-^ΔCt计算。数据分析使用GraphPad Prism 5软件，检验方法为two-tailed Student's test。最终结果用平均值±SD(标准差)表示。
结果表明，血清和血浆样本都可用于S-Poly(T)Plus法检测miRNA的模板，但是血浆中miRNA相对表达量显著高于血清，再次验证miRNA在血浆中的表达量显著高于血清(图6)。
实施例2 本发明的Direct S-Poly(T)Plus(DSPP)方法中不同裂解方案的效果比较
在Direct S-Poly(T)Plus方法中，可选用以下六种处理方式中的任一种实现miRNA从蛋白复合体中裂解出来：
①裂解体系：20ul裂解液、20ul样本；裂解条件：75℃保持5分钟；
②裂解体系：20ul RNase-free water、1ul蛋白酶K、20ul样 本,；裂解条件：50℃处理20分钟,然后95℃保持5分钟；
③裂解体系：20ul裂解液、1ul蛋白酶K、20ul样本；裂解条件：50℃处理20分钟,然后95℃保持5分钟；
④裂解体系：20ul 2×lysis buffer、20ul样本；裂解条件：75℃保持5分钟；
⑤裂解体系：20ul 2×lysis buffer、1ul蛋白酶K、20ul样本,；裂解条件：50℃处理20分钟,然后95℃保持5分钟；
⑥裂解体系：10ul 2×lysis buffer、10ul裂解液、1ul蛋白酶K、20ul样本；裂解条件：50℃处理20分钟,然后95℃保持5分钟。
上述处理方式中所述的裂解液包括以下终浓度的组分：2.5％的tween-20,50mM Tris和1mM EDTA；所述2×lysis buffer包含以下终浓度的组分：100mmol/lTris-HCl、300mmol/l NaCl、20mmol/l MgCl 2；pH为8.0；所述蛋白酶K的终浓度为15U/mL。
其他操作同实施例1。
实验结果显示，在上述方案中，单独使用tween 20(裂解液的主要功能成分，详见参考文献Zhang Q，Oncotarget，2016，7(16):21865–21874)、蛋白酶K或者二者同时使用的效果都差强人意(分别对应方案①，②，③)。在方案⑤中，使用2×lysis buffer和蛋白酶K的组合，miRNA的Ct值最小，与方案③相比，降低了0.8～6.8。方案⑤和⑥对比实验，表明可能tween 20在裂解miRNA包裹蛋白复合物的同时，也能对poly(A)/RT反应造成不利影响(图2)。因此， 方案⑤在本发明中被推荐为最优方案，裂解反应血浆用量为20～50ul。
实施例3 Direct S-Poly(T)Plus方法中一步法和两步法灵敏度对比
在之前的发明S-Poly(T)Plus方法中(专利申请号：201510558101.5)，以纯化的RNA为模板，一步法灵敏度比两步法大大提高。两步法即miRNA Poly(A)加尾完成后再进行逆转录；一步法即miRNA的Poly(A)加尾和逆转录在同一反应中进行。在本次发明中，以粗提RNA为模板，操作同实施例1，两步法与一步法的灵敏度再次比较。如图3所示，在Direct S-Poly(T)Plus方法中，本发明方案使一步法的灵敏度比其两步法提高了2.5～52倍(1.7～5.7个Ct值差距)(图3)。
实施例4 miRNA的直接荧光定量PCR扩增技术体系起始粗提RNA加入比例效果对比
粗提RNA可能含有一些抑制Poly(A)加尾和逆转录酶活性的成分，因此在Direct S-Poly(T)Plus方法中粗提RNA加入的起始量对方法灵敏度的存在一定影响，采用不同的粗提RNA起始量，试验操作同实施例1,试验结果如图4，可以看出，当粗提RNA的起始量体积百分数从0.5％增长至40％时，miRNA的Ct值线性降低。但是当粗提RNA加入的比例升高至60％和75％时，miRNA的Ct值出现了回复升高。在本发明中，40％的粗提RNA起始量被推荐为最佳比例。
实施例5、热启动DNA聚合酶在Direct S-Poly(T)Plus方法中 的作用测试
在Direct S-Poly(T)Plus方法体系中，没有RNA的纯化，可能会引入一些基因组DNA的污染，因此在qPCR中，更容易出现与基因组DNA的错配。减少非特异性扩增的一个有效方法是热启动，即在热循环开始之前防止或者减少DNA的合成。本实施例对Direct S-Poly(T)Plus方法中PCR部分所用的普通DNA聚合酶和热启动DNA聚合酶做了对比分析。本实施例中采取了一种有效的形成热启动的方法，即Taq酶抗体，抗体与DNA聚合酶结合，在热循环开始之前，酶活不会被启动。本实施例中采用的热启动DNA聚合酶为Hotstart Alpha Taq Polymerase，具体制备方法为Alpha Taq Polymerase(VitaNavi,St.Louis USA)与Taq Antibody(菲鹏公司，深圳)等体积混合，室温放置6小时。在图7和图8中可见，使用热启动酶可以有效减少非特异性扩增。
实施例6、Hotstart Alpha Taq Polymerase的用量对Direct S-Poly(T)Plus方法扩增效率的影响
本实施例对Hotstart Alpha Taq Polymerase的酶量对miRNA的直接扩增效率做了分析。从图9中可以看出，Hotstart Alpha Taq Polymerase的活性非常高，20ulPCR体系中0.0125uL的酶量即可满足扩增要求。
实施例7、Hotstart Alpha Taq Polymerase的用量对Direct S-Poly(T)Plus方法中出现的非特异性扩增的影响
本实施例探索了不同用量Hotstart Alpha Taq Polymerase对非 特异性扩增的影响，从图10中可以看出，20uLPCR体系加入0.4uL Hotstart Alpha Taq Polymerase会造成一定的非特异性扩增；如果酶量降低到0.0125uL(如图11)，非特异性扩增会得到很好地抑制。
实施例8、Direct S-Poly(T)Plus方法检测血浆miRNA的线性梯度范围
本实施例对Direct S-Poly(T)Plus方法检测血浆miRNA的线性梯度范围进行了分析。将血清RNA进行4倍梯度稀释(总RNA使用量对应的初始血浆的用量为0.1-0.0004ul)，然后进行检测。从图12看出，Direct S-Poly(T)Plus方法检测血浆miRNA(hsa-miR-451a,hsa-miR-21-5p,hsa-miR-126-3p,hsa-miR-92a-3p,hsa-miR-210-3p,hsa-miR-27b-3p,hsa-miR-103a-3p和hsa-miR-92a-3p)都具有较好的线性相关系数R2(0.9139-0.9988)。因此，Direct S-Poly(T)Plus方法检测血浆miRNA具有良好的线性关系和较宽的动态范围。
实施例9、Direct S-Poly(T)Plus方法与其他方法比较
在本实施例中Direct S-Poly(T)Plus方法将与最为流行的Stem-loop方法和对比例1中的S-Poly(T)Plus方法做比较。其中Stem-loop和S-Poly(T)Plus的方法是以纯化的RNA做模板。S-Poly(T)Plus方法同实施例1，Stem-loop方法操作方法则按照试剂盒TaqMan microRNA assay kit(Applied Biosystems)说明书。
本实施例中，共用三种miRNA检测方法检测六个miRNA，即hsa-miR-140-5p,hsa-miR-124a-3p,hsa-miR-16-5p,hsa-miR-93-5p, hsa-miR-25-3p和hsa-miR-106-5p。如图13所示，除去hsa-miR-16-5p(25.43)和hsa-miR-93-5p(27.78)的Ct值在S-Poly(T)Plus方法中略小，其余的miRNA Ct值均在Direct S-Poly(T)Plus方法中最小。Direct S-Poly(T)Plus方法比stem-loop方法灵敏度高出7-342倍(2.8-8.4个Ct值)。
实施例10、Direct S-Poly(T)Plus方法分析结直肠癌病人miRNA表达谱
在本实施例中，用Direct S-Poly(T)Plus方法做了六个miRNA的单样本验证，内参hsa-miR-93-5p为归一化标准，使用血浆样本来自于30个健康志愿者和30个结直肠癌病人。从图14可以看出，hsa-miR-22-3p,hsa-miR-423-5p,hsa-miR-144-3p和hsa-miR-451a在单个样本验证中表达量显著上调，hsa-miR-30b-5p表达量显著下调，hsa-miR148a-3p表达量没有显著变化。
对比例2、S-Poly(T)Plus方法分析结直肠癌病人miRNA表达谱
在本实施例中，为了验证Direct S-Poly(T)Plus方法得出的结论，再次使用S-Poly(T)Plus进行六个miRNA的单样本验证，外参cel-miR-54为归一化标准，使用血浆样本同实施例10。从图15可以看出，六个miRNA的表达趋势与Direct S-Poly(T)Plus方法得到的结果一致。因此，我们可以得出结论，Direct S-Poly(T)Plus方法是稳定可靠的，并且hsa-miR-22-3p,hsa-miR-423-5p,hsa-miR-144-3p,hsa-miR-451a和hsa-miR-30b-5p可以作为结直肠癌潜在的生物标记物。
本发明以S-Poly(T)Plus技术为基础，介绍一种灵敏但无需进行RNA提取的miRNA检测方法，即miRNA的直接荧光定量PCR扩增技术(Direct S-Poly(T)Plus,简称DSPP)。在Direct S-Poly(T)Plus方法中，miRNA首先要从蛋白复合物中释放出来，得到粗提的RNA；然后基于S-Poly(T)Plus方法，粗提RNA在同一反应体系中同时加尾和逆转录。忽略操作时间，用本发明中的Direct S-Poly(T)Plus方法，cDNA可以在95分钟内制备完成，加上qPCR时间，140分钟可以完成整个miRNA检测流程。从48个样品中检测1个miRNA，仅需3个小时即可完成整个操作过程，而提取核酸的方法至少需要一天时间。此项Direct S-Poly(T)Plus技术，将会极大地简化检测流程、降低成本，更有力的推动循环miRNA肿瘤标志物早日进入临床应用。
以上所述实施例仅表达了本发明的几种实施方式，其描述较为具体和详细，但并不能因此而理解为对本发明专利范围的限制。应当指出的是，对于本领域的普通技术人员来说，在不脱离本发明构思的前提下，还可以做出若干变形和改进，这些都属于本发明的保护范围。因此，本发明专利的保护范围应以所附权利要求为准。

Claims (10)

  1. 一种直接定量检测循环miRNA的RT-qPCR方法，其特征在于，所述方法，包含以下步骤：
    S1、裂解离心：利用裂解用试剂将样本中的蛋白复合体中充分裂解，使miRNA从样本中释放出来；短暂离心后，所得上清液即为粗提RNA；
    S2、加尾逆转录：将所述步骤S1中所获得的粗提RNA进行加Poly(A)尾及S-Poly(T)特异性逆转录；
    S3、RT-qPCR定量检测：以步骤S2中获得的逆转录产物cDNA为模板进行RT-qPCR定量检测。
  2. 根据权利要求1所述的检测方法，其特征在于，所述步骤S1中裂解用试剂包括组分：20ul 2×lysis buffer、1ul蛋白酶K，所述裂解用试剂对应处理20ul样本。
  3. 根据权利要求2所述的检测方法，其特征在于，所述2×lysis buffer包含以下终浓度的组分：100mM Tris-HCl、300mM NaCl、20mM MgCl 2；pH为8.0。
  4. 根据权利要求2所述的检测方法，其特征在于，所述蛋白酶K的终浓度为15U/mL。
  5. 根据权利要求1所述的检测方法，其特征在于，所述步骤S1中裂解用试剂的反应条件为50℃处理20分钟，然后95℃保持5分钟。
  6. 根据权利要求1所述的检测方法，其特征在于，所述步骤S1中的离心条件为：10，000～14，000g，4℃条件下离心5～15分钟。
  7. 根据权利要求1所述检测方法，其特征在于，所述步骤S2中加尾逆转录反应体系中所加入粗提RNA模板的体积百分比为5～75％。
  8. 根据权利要求1所述的检测方法，其特征在于，所述步骤S2中加尾逆转录的反应体系包括：0.5-7.5uL上清模板、1±0.2μL的0.5μmol/L RT primer、1±0.2U的PolyA Polymerase、100±20U的MMLV、2.375-0.625uL的reaction buffer、RNase-free Water补足至10μL；加尾逆转录的反应条件为：37～42℃保温50～70min，74～76℃保温3～7min以灭活酶，然后迅速置于冰上，静置2min以终止灭活。
  9. 根据权利要求1所述的检测方法，其特征在于，所述步骤S3中real-time PCR反应体系为：4×qPCR reaction Buffer 5μL、1μmol/L的Forward Primer 4μL、10μmol/L的universal reverse primer0.4μL、10μmol/L的universal Taqman probe0.5μL、100×ROX Rerference Dye0.2μL、Hotstart Alpha Taq Polymerase0.0125μL、cDNA 0.5μL、RNase-free Water加至20μL；反应条件为：预变性95℃5分钟，变性95℃10s，退火60℃40s，40个循环。
  10. 根据权利要求1-10任意一项所述的检测方法，其特征在于，所述样本为血浆、血清、尿液、眼泪、乳汁、唾液、痰液或粪便抽提上清。
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