WO2016015686A1 - 一种利用扩增dna片段长度多态性定量测定短链rna的方法 - Google Patents

一种利用扩增dna片段长度多态性定量测定短链rna的方法 Download PDF

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WO2016015686A1
WO2016015686A1 PCT/CN2015/089566 CN2015089566W WO2016015686A1 WO 2016015686 A1 WO2016015686 A1 WO 2016015686A1 CN 2015089566 W CN2015089566 W CN 2015089566W WO 2016015686 A1 WO2016015686 A1 WO 2016015686A1
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rna
short
mirna
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dna fragment
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徐凯
唐放
张耀艺
冯梓浩
杨宇
吴秀锦
张菲菲
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成都诺恩生物科技有限公司
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Definitions

  • the invention relates to the field of molecular biotechnology, in particular to a method for quantitatively determining short-chain RNA by using amplified DNA fragment length polymorphism.
  • MicroRNAs are a class of short single-stranded small-molecule ribonucleic acids that regulate gene expression and consist of 22 or so nucleotides (nt). miRNAs are involved in various activities of cells, affecting and participating in maintaining the physiological and differentiation state of cells. The changes in the expression levels of specific miRNAs can also reflect the physiological and pathological characteristics of organisms, indicating their potential as biomarkers for identifying various diseases. For example, miR-122, which is characteristic of hepatocytes, changes in the blood level to reveal the damage of the liver. miR-122 is unique to liver cells and has 130,000 in each human liver cell, which has the specificity and sensitivity that are currently lacking in biomarkers for liver damage identification.
  • miRNAs Although the role of miRNAs in these lesions remains to be determined, the mechanism needs to be further revealed, but this has become one of the most important directions in miRNA research.
  • the objectivity, sensitivity and standardization of miRNA quantitative detection methods are the bottleneck and key to push miRNA research results into clinical disease diagnosis applications.
  • miRNAs It is known that humans have more than one thousand miRNAs, and the dynamic range of miRNA expression associated with various specific physiological and pathological phenomena ranges greatly from single digits to tens of thousands, plus miRNAs have ultra-short lengths and similar The characteristics of sequence, secondary conformation, terminal modification and length difference pose great challenges for the purification and accurate quantification of miRNA.
  • RNA/DNA hybrid technology such as NanoString nCounter and nanopore technology
  • the most sensitive NanoString nCounter method has a lower limit of 1 amol, that is, a level of about 6 ⁇ 10 5 molecules or more, and the measurement limit of miR-seq and microArray is 0.1 fmol. 6x10 levels above 7 molecules clearly do not meet the needs of most molecular diagnostics.
  • the miR-seq method does not depend on miRNA sequences for miRNA identification, and can have an irreplaceable role in the discovery of new miRNA species. Moreover, it can count the relative readings of miRNAs, and many studies have used it to explore many pathological diagnostic applications and found many beneficial signs.
  • miR-seq requires the preparation of a suitable cDNA library using RNA Ligase or DNA Ligase, which can be detected by PCR and then detected on the machine for relative quantification.
  • RNA Ligase or DNA Ligase which can be detected by PCR and then detected on the machine for relative quantification.
  • the lengthy sample preparation process and the use of various modified enzymes destroy the linear correlation between the number of targets to be tested and the detected readings, and affect the objectivity of miRNA determination. Therefore, if there is no better preparation method, miR-seq cannot be used as a method for quantitative determination of miRNA.
  • RT-qPCR Reverse transcription real-time fluorescent PCR quantitative technology
  • Traditional RT-qPCR assays are long-chain RNAs, such as mRNA, and the primers used are typically more than 20 nt in length. However, the total length of mature miRNA is only about 22 nt, and the RT-qPCR for determining miRNA must be specially modified to make it suitable for qPCR.
  • the RT of the stem loop primer as the miRNA was also applied to the quantitative determination of miRNA.
  • Real-time fluorescent PCR quantitative method represented by reverse transcription-qPCR method using TaqMan probe and stem-loop primer has the ability to measure single-copy miRNA molecules, and has been widely used for mechanism research, disease diagnosis, treatment prognosis, etc. of miRNA. In the study. Because it is the most sensitive, objective, and easy to apply method of all methods, it is the preferred method to verify the miRNA expression profile obtained from the microArray, miR-seq method.
  • the minimum limit of the method is 1 amol, which is 6x10 5 copies, which does not meet the actual needs of clinical diagnosis.
  • the hybridization signal is not amplified, and the thermodynamic balance of hybridization is restricted.
  • the sensitivity of the method is difficult to break.
  • the lower limit of amol Another possible difference is that the miRNA labeling reaction is limited by the miRNA end sequence, secondary conformation, and the labeling method used. It is difficult to label all kinds of miRNAs in equal proportions, which brings about the measurement of different miRNAs. Discriminatory bias.
  • the randomness of the influence increases the difficulty of standardization. Therefore, in the strict sense, absolute quantification is impossible to achieve with existing measurement methods.
  • the impurities contained in the RNA purified from different individual sera can inhibit or even promote the RT reaction and PCR amplification efficiency, and increase the difficulty of quantitative determination using traditional RT-qPCR method. .
  • the treatment and purification of the sample generally use a protein denaturant extraction method such as guanidin hydrochloride to obtain a highly pure RNA, but since the average size of the miRNA is only 22 nt, a high concentration of ethanol is required for long-term precipitation to avoid loss, so that purity and The contradiction of the yield is more prominent, and the purification method using the affinity resin column generally ensures the purity of the RNA but still needs to prove the integrity of the purified RNA, that is, the loss of miRNA and the loss of selectivity.
  • Pathological specimens obtained from patients are usually small and not reproducible, and therefore, these problems have plagued efforts to push miRNA quantitative detection technology into clinical applications.
  • An object of the present invention is to provide a novel method for improving the sensitivity, accuracy and standardization of short-strand RNA quantitative measurement in view of the above problems.
  • the technical scheme adopted by the present invention is as follows: a method for quantitatively determining miRNA by using amplified DNA fragment length polymorphism, comprising the following steps:
  • At least two synthetic small RNAs having no natural homologous sequence compared with the short-chain RNA to be tested are used as internal standards of the assay, and the synthetic small RNAs as internal standards are mixed in different molecular numbers to form dynamic small RNA standard molecules. gradient;
  • the same amount of the dynamic small RNA standard is mixed with the short-chain RNA to be tested, and the fluorescence is quantitatively analyzed by RNA reverse transcription, cDNA tailing, PCR synchronous amplification and PCR product DNA length polymorphism fragments.
  • the relative ratio of the fluorescence intensity of the DNA fragment produced by the amplification of the short-chain RNA to be tested to the standard small RNA standard fluorescence intensity gradient is obtained, thereby achieving relative quantification of the short-chain RNA to be tested.
  • a synthetic small RNA having a different molecular number as a dynamic small RNA standard is first mixed with a short-chain RNA to be tested in the sample, and synchronous detection is performed in the same reaction, and the molecular standard amount measured by the synchronization is a dynamic quantitative standard. Scale, obtain the relative peaks of the short-chain RNA to be tested.
  • the synthetic small RNA as an internal standard requires no natural homologous sequence to the short-chain RNA to be tested. Impurities or factors affecting the reaction have the same degree of influence on the short-chain RNA to be tested and the dynamic small RNA standard molecules in the sample.
  • the short-strand RNA reference sample synthesized by using the short-stranded RNA sequence as a template is short.
  • the sequence of the stranded RNA reference is the same as the sequence of the short-stranded RNA to be tested, and the number of different molecules, the relative number of molecules obtained in the above-mentioned dynamic small RNA standard is the relative ratio of the gradient, and the relative intensity correction curve is utilized.
  • the relative intensity ratio of the strand RNA is calculated from the calibration curve to calculate the absolute molecular number of the short-chain RNA to be tested in the sample.
  • the absolute number of RNAs to be tested is obtained by comparing the calibration curves of known amounts of synthetic target RNA with relative measurements of the same small molecule RNA standard to obtain an absolute quantitative result of the RNA to be tested.
  • the short-chain RNA to be tested is a miRNA or an siRNA.
  • the method of the present invention can be used for determining the type (qualitative) and content (quantitative) of miRNA, siRNA and RNA in various biological samples or purified RNA, and the siRNA has the same physical and chemical characteristics as the miRNA, and the above method is also applicable to Qualitative and quantitative determination of siRNA.
  • the invention is also applicable to the identification of one or several smaller sequences in RNA, thereby achieving the qualitative and quantitative determination of various RNAs, and is particularly suitable for short-chain RNAs such as miRNAs.
  • miRNA signatures are the expression levels of several to dozens of miRNAs associated with a life phenomenon in a particular sample, and their changes can reveal relevant physiological or pathological changes.
  • the implementation of the present invention can conveniently, rapidly, sensitively and accurately identify miRNA fingerprints in one or several reactions, and contribute to the research of miRNA fingerprinting and its application in clinical diagnosis.
  • the present invention designs a method for performing absolute quantitative detection of RNA, particularly miRNA, by relative quantitative analysis of DNA fragment length polymorphism fluorescence (miRNA-derived Fragment Length Polymorphism Assay, referred to as miRFLP quantitative assay in the present invention).
  • miRFLP quantitative assay uses three synthetic small RNAs without natural homologous sequences as internal standards for the assay, and the three internal standard small RNAs are mixed with different molecular numbers to form a dynamic small RNA standard molecular gradient, and then an equal amount of dynamics.
  • the small RNA standard is mixed with the short-chain RNA to be tested, and the DNA generated by the short-stranded RNA to be detected is measured in four steps including RNA reverse transcription, cDNA tailing, PCR synchronous amplification, and PCR fragment length polymorphism analysis.
  • the conversion curve of the absolute number of short-chain RNA can be plotted, thereby calculating the absolute content of the short-chain RNA to be tested. the amount.
  • the primer used in the reverse transcription of the RNA is an omega primer.
  • the omega primers of the present invention are, inter alia, the omega primers disclosed in PCT Patent Application Serial No. PCT/CN2013/070525.
  • the omega primer serves as a primer probe for the quantitative analysis reaction of the miRFLP of the present invention.
  • the omega primer contains a highly stable secondary stem-loop structure that avoids the formation of primer-dimers, improves end-initiation accuracy, and effectively separates primers into probe and coding regions.
  • Different primers can be length-encoded by adding or subtracting the number of bases in the coding region, and various miRNAs can be converted into cDNA fragments of different lengths in the same reverse transcription reaction, and fluorescent fragments can be simultaneously amplified by PCR. Length polymorphism analysis solves the key technical problems of detecting multiple miRNAs in a single reaction.
  • the miRFLP reaction consists of four steps: miRNA reverse transcription, cDNA tailing, PCR simultaneous amplification and PCR fragment length polymorphism analysis, as shown in Figure 1.
  • the first step in the reaction is to hybridize the miRNA and the omega primer, and the miRNA forms a pair with the complementary probe, and then synthesizes the cDNA with the unpaired miRNA 3' end as a template using reverse transcriptase.
  • the newly synthesized cDNA was hybridized with a 3' oligonucleotide primer containing a common PCR target and the DNA strander was used to complement the single-stranded gap after DNA pairing starting from the 3' end of the cDNA.
  • the correctly assembled cDNA has the same 5' and 3' end sequences, and can be synchronously amplified by a pair of fluorescent PCR primers.
  • the amplified products are separated by capillary gel electrophoresis and the PCR fragments of different lengths are determined. Fluorescence intensity, complete miRFLP analysis map.
  • the primer used in the reverse transcription of the RNA is a stem-loop primer.
  • the stem loop primer is a length-coded stem loop primer.
  • the length encoding method comprises: adding a different number of bases between the PCR target site of the stem-loop primer and the probe sequence, and adjusting the base sequence at the 5′ end of the primer. Maintain the secondary structure of the stem ring unchanged.
  • a primer probe of the stem-loop structure (Applied Biosystems, Inc, as mentioned in PCT/CN2013/070525) simultaneously converts a plurality of small RNAs into cDNA quantitatively, and tails up and reacts with a cDNA product similar to the above miRFLP reaction pathway. Fluorescence fragment length polymorphism analysis was performed by PCR simultaneous amplification. Experimental results demonstrate that stem loop primers can also be used in the design of the present invention as long as a variable length coding region is added, and can be used interchangeably or simultaneously with omega primers when determining a limited number of miRNA targets.
  • the calibration curve conforms to the logarithmic regression method and is expressed as: aX b , wherein a and b are constant terms, which are determined by actual measurement values of the different molecular numbers of the synthetic small RNA.
  • the number of synthetic small RNAs having no natural homologous sequence compared with the short-chain RNA to be tested is three. Since the fluorescence intensity and the number of fluorescent substances are not linear, at least three kinds of regressions are needed to determine the regression of the quadratic equation, and the three synthetic small RNAs can meet the requirements of quantitative determination and the detection process is also economical.
  • the realization of the intermediate product and the amplification of the signal in each step of the miRFLP quantitative assay are performed in a linear or near-linear quantitative manner, so the fluorescence intensity of the amplified DNA fragment is linearly and quantitatively related to the number of molecules of the target miRNA. .
  • the response curve between the fluorescence intensity of the instrument probe and the amount of the fluorescent substance is suitable for the conversion between the measured fluorescence intensity of the target to be measured and the number of molecules thereof.
  • the sample was analyzed by fluorescence quantitative analysis with ABI3730xl DNA analyzer.
  • the dilution ratio and the measured fluorescence intensity were determined.
  • the correlation is consistent with the regression relationship of the quadratic equation in the range of 25–25000FU, and the average value of R 2 is greater than 0.9999.
  • the dynamic miRNA standard used in the method and the amplification of the sample target fluorescence signal are synchronized in a nearly linear quantitative manner, and the unary quadratic regression equation is applicable to the calculation of the relative fluorescence intensity of the miRNA to be tested on the dynamic miRNA standard.
  • Modern DNA sequencers have extremely high sensitivity and dynamic detection range (ABI 3730 DNA Analyzer: 5-31000FU). Different manufacturers or different types of DNA fluorescence quantitative analyzers can use the above methods to calibrate the applicable fluorescence response curve and range, and select the most suitable and most accurate regression method.
  • the measured miRNA value is not the absolute molecular number of the miRNA, but the ratio of the miRNA to be tested on the fluorescence intensity gradient of the dynamic miRNA standard, and the ratio of the ratio reflects the test.
  • a calibration curve of the relative ratio of the miRNA to be tested to the number of miRNA molecules to be tested can be made, and the relative ratio of the miRNA to be tested is converted into its miRNA. Absolute copy number.
  • Synthetic miRNA reference dilutions with two or three dilutions can also be used to accurately measure the range of methods and approximate error margins at each concentration point, which can help improve the confidence and standardization of the resulting data analysis results.
  • the invention is applicable to the simultaneous identification of multiple miRNAs, and a plurality of miRNA synthesis reference materials can also be used in combination, which greatly reduces the number of reactions required.
  • the beneficial effects of the present invention are that the miRFLP quantitative analysis method of the present invention has at least the following advantages compared with the current method:
  • Absolute quantification Absolute quantification of miRNAs to be tested by benefiting from dynamic small RNA standard molecules formed by reaction with sample RNA in the same reaction tube, eliminating complex samples and interference with RNA purity determination;
  • miRNAs with only one base difference or the same family can be identified;
  • the amount of sample for the single-copy miRNA assay is 0.1 ⁇ g-1ng total RNA
  • Controlled synthetic miRNA standard reference is used to ensure the standardization of methods by ensuring repeatability and comparability between operators and laboratories.
  • RNA extract The expression level of miRNA can be accurately and objectively measured directly from patient samples such as serum and plasma, and the results are reliable and reliable.
  • FIGS. 1A and 1B are schematic diagrams showing the reaction flow and measurement of the miRFLP quantitative analysis method
  • 2 is a fluorescence analysis of a DNA fragment length after a 25-fold dilution of a randomly selected fluorescent PCR product having a plurality of hetero peaks;
  • Figure 3 is a graph showing the relationship between the fluorescence measurement value and the dilution factor of the DNA fragments of 62.00 nt and 91.22 nt (B) at different dilutions in accordance with the quadratic equation regression curve;
  • 4A and 4B are miRFLP analysis maps of dynamic miRNA standards and different concentrations of miRNA
  • std 1", “std 2", and “std 3” respectively represent fluorescent DNA fragments of dynamic miRNA standards (std 1, std 2, and std 3), "1", “2", “ 3” and “4" represent reaction 1, reaction 2, reaction 3, reaction 4, respectively, "a” represents a fluorescent DNA fragment of miR-92a, and "b” represents a fluorescent DNA fragment of miR-92b, sitting vertically Marked as fluorescence intensity;
  • 5A and 5B are miRFLP assay profiles and standard curves of different molecular numbers miR-92a and miR-92b;
  • FIG. 5A and Fig. 5B are blank control assay containing only dynamic miRNA standards, and "2-10” is added with 2.5x10 5 to 244 synthetic miR-92a molecules and an equal amount of miR-92b, respectively.
  • FIG. 5B is a point correspondence diagram of different miRNA copy numbers and their relative fluorescence intensities;
  • Figure 6 is a diagram showing the repeatability of the miR-25 family member correction curve and the results of the measurement range by the miRFLP method
  • Figure 7 is a graph showing the results of a Le-7 family miRFLP quantitative analysis profile for a single Let-7 member
  • Figure 8 is a measurement result of miR-92b and miR-25 by a miRFLP analysis method using a stem-loop primer
  • Figure 9 shows a stem-loop primer encoded by the number of bases
  • FIG. 9 A: a stem-loop primer designed to detect Standard 1 RNA, and 4 bases (boxes) were placed between the 5'-end PCR target (arrow) and the probe as a label.
  • Stem ring dG -13.71.
  • Stem ring dG -13.09.
  • an omega primer is taken as an example to specifically describe a reaction flow and a measurement principle of the miRFLP quantitative analysis method.
  • the miRFLP reaction consists of four steps: miRNA reverse transcription, cDNA tailing, PCR simultaneous amplification and PCR fragment length polymorphism analysis, as shown in Figure 1A and Figure 1B.
  • the first step in the reaction is to hybridize the miRNA and the omega-gamma primer, and the miRNA forms a pair with the complementary probe, and then synthesizes the cDNA with the unpaired miRNA 3' end as a template using reverse transcriptase. After removal of the RNA, the newly synthesized cDNA was hybridized with a 3' oligonucleotide primer containing a common PCR target and the DNA strander was used to complement the single-stranded gap after DNA pairing starting from the 3' end of the cDNA.
  • the correctly assembled cDNA possesses the same 5' and 3' end sequences, and can be synchronously amplified by a pair of fluorescent PCR primers, and the amplified product passes through capillary gel electrophoresis.
  • the fluorescence intensity of PCR fragments of different lengths was determined after swimming separation, and the miRFLP analysis map was completed.
  • Figure 1B is the final step of the quantitative analysis of the whole miRFLP.
  • the fluorescence intensity measured by the fluorescence quantitative analyzer is proportional to the amount of the fluorescent substance to be tested. This relationship is related to the fluorescent probe configured by the instrument, that is, different fluorescent probes have different fluorescence response curves.
  • the fluorescence intensity measured by ABI's Prizma 310 DNA sequencer is linearly quantitatively related to the amount of fluorescent substance to be measured in the range of 5-7000FU, and changed to a parabola when the fluorescence intensity value is greater than 7000FU. Fluorescence probes of different instruments have different response characteristics, which can affect the regression relationship between the measured fluorescence intensity and the amount of fluorescence to be measured.
  • the fluorescence response curve of this example for the ABI 3730xl DNA Analyzer was calibrated with a 2-fold serial dilution of the PCR product.
  • a random fluorescent PCR product with multiple different degrees and a large change in fluorescence intensity was randomly selected and diluted with 25x, 50, 100, 200 and 400 times in 1xTE and analyzed by ABI 3730xl DNA analyzer.
  • Figure 2 is a fluorescence scan of the 25-fold diluted sample of the PCR product, and 11 DNA fragments with peak values between 253 and 25000FU were selected, and their fluorescence readings at different dilutions were counted (see Table 1), via IBM SPSS.
  • Statistics 20 statistical software optimizes the regression curve for the correspondence between dilution factor and fluorescence intensity measurement.
  • the quadratic equation regression curve is suitable for describing the relationship between fluorescence intensity and dilution factor of all slices, and the goodness of fit of regression.
  • the R square values are all greater than 0.999. Therefore, the correspondence between the fluorescence intensity of the DNA fragment and the amount of the fluorescent substance measured by the ABI 3730xl DNA analyzer can be accurately calculated using a one-dimensional quadratic regression curve instead of a simple linear quantitative relationship. Repeated experiments have shown that the absolute values of the same samples in different batches can be different, but the regression model of this one-dimensional quadratic equation is not affected.
  • Figure 3 shows the regression curves after serial dilution of the low abundance fragment (A: 30FU-312FU) and the high abundance fragment (B: 2052FU-24949FU). Different manufacturers or different types of DNA fluorescence quantitative analyzers can use the above methods to calibrate the applicable fluorescence response curve and range, and select the most suitable and most accurate regression method.
  • Table 1 Fluorescence values measured at 25, 50, 100, 200, and 400-fold dilutions of the PCR product of Figure 2 using the ABI 3730xl DNA Analyzer at various dilutions and unary by IBM SPSS Statistics 20 statistical software Goodness of fit R square after quadratic regression analysis:
  • the miRFLP assay mixes the miRNA to be tested with the dynamic miRNA standard, and performs miRNA reverse transcription, cDNA tail modification and fluorescent PCR simultaneous amplification. Finally, the DNA fragment length and fluorescence quantitative analysis are performed by DNA sequencer. Specifically, 4 ⁇ l of the hybrid mother liquor was first prepared: 2 ⁇ l of 5 ⁇ RT buffer, 1 ⁇ l of 10 mM MgSO 4 , and 1 ⁇ l of a mixture of dynamic miRNA standards, and the mixture included “standard 1 RNA” having a molecular number of 3 ⁇ 10 6 (ie “std1” in FIG. 4A”.
  • RNA sequence of the dynamic miRNA standard is shown in Table 2, and the sequence search by the Sangers miRbase version 20 database proves that there is no homologous sequence).
  • reverse transcriptase mixture (1 ⁇ l of MMTV reverse transcriptase, 0.5 ⁇ l of dNTP, 0.5 ⁇ l of water, Takara) was added, mixed, and incubated at 25 C for 30 minutes, 37 ° C for 10 minutes, and denatured at 85 ° for 5 minutes.
  • the PCR product was diluted at a dilution of 1:20 or 1:50, and then subjected to fluorescence fragment length polymorphism analysis using an ABI 3730xl DNA analyzer.
  • the miRFLP analysis map results are shown in Figures 4A and 4B;
  • reaction 1 is the result of a blank control containing only dynamic miRNA molecular standards; 2.5x10 5 miR-92a molecules and an equivalent amount of miR-92b are added to reaction 2, and in Figure 4B, reaction 3 3.1x10 4 miR-92a and an equal amount of miR-92b were added as targets to be tested, and 78 pg of H1299 total RNA was added to reaction 4.
  • DNA fragments of different lengths represent different miRNA molecules (the value above the peak is the fragment length, nt), and the fluorescence intensity reflects the relative amount of the miRNA (below the peak note).
  • the value is the fluorescence intensity, FU), and the fluorescence intensity measured by different dynamic miRNA standards in the same reaction forms a fluorescence intensity gradient corresponding to the number of molecules.
  • the absolute number of various dynamic miRNA standards placed in proportion to different reactions is consistent, but for various reasons, such as the different properties of operations, instruments, consumables, reagents, and measurement conditions, the absolute determination of fluorescence intensity is affected.
  • the concentration of the preservation solution for RNA preservation in the reaction 2 is higher than that of the reaction 3, which affects the efficiency of the RT or PCR reaction, and causes the fluctuation of the dynamic miRNA standard fluorescence intensity gradient. Since the target to be tested has been mixed with the dynamic miRNA standard before reaction, the factors affecting the efficiency of the reaction are the same as the dynamic miRNA standard.
  • the relative ratio of the fluorescence intensity of the target to be measured in the dynamic standard fluorescence intensity gradient can be It is used to objectively measure the molecular ratio between the target to be tested and the dynamic miRNA standard.
  • Table 2 miRFLP quantitative assay for the determination of miM-92a and miR-92b omega primers
  • the base sequences are SEQ ID NO. 1, SEQ ID NO. 2, respectively;
  • the base sequence of the Omega-Miga primer used is SEQ ID NO. 3;
  • the base sequence of the 3' oligonucleotide-adaptive primer used ie, "3' Adapter” in Table 2
  • SEQ ID NO. 4 SEQ ID NO. 5
  • dynamic miRNA standard Std 1,2,3
  • the base sequences are SEQ ID NO. 6, SEQ ID NO. 7, and SEQ ID NO. 8, respectively;
  • the base sequences of the omega miga primers used respectively are SEQ ID NO.9, SEQ ID NO. 10, SEQ ID NO.
  • the base sequence of the 3' oligonucleotide-adaptive primer used is SEQ ID NO. 12) and its composition: Table: dG, dC
  • Table: dG, dC The average molecular weight of dA and dT (308.95 Daltons) is 1 nt, and the molecular weight of 5' fluorophore is 474.5.
  • the miRFLP quantitative analysis reaction performs reverse transcription, modification and amplification reactions on small RNAs in the same reaction in a linear proportional manner.
  • the dynamic standard fluorescence intensity gradient can exclude the influence of external factors on the measurement, but the Tm of the probe.
  • the difference in length and conformation of the PCR fragment can still affect the fluorescence intensity of the DNA fragment.
  • the influence of the latter two can be reduced to negligible by design and optimization of reaction conditions, but the influence of the probe Tm on the fluorescence intensity produced by the target cannot be ignored. Therefore, it is necessary to make a calibration curve between the number of molecules and the relative fluorescence intensity with a known number of targets to be measured in order to convert the relative fluorescence intensity of the target to be measured into the actual number of molecules of the target to be tested.
  • a serially diluted synthetic miRNA as the target to be measured, a conversion curve of relative fluorescence intensity to the number of target molecules can be obtained.
  • the synthetic miR-92a and miR-92b nucleic acid small molecules which are mixed in equal amounts are diluted to a target of 2 times dilution, and the measurement conditions are determined according to the miRFLP analysis method of miR-92 in Example 3.
  • the experimental group was assayed for 3.12 ng, 0.312 ng, 0.0312 ng of A549 cellular RNA, 0.222 ng of Hela cellular RNA, and 0.25 ng of H1299 cellular RNA. Three replicate experiments were set for each assay reaction to determine the error range of the assay.
  • FIG. 5A shows the number of molecules of miR-92a and miR-92b in each reaction.
  • FIG. 5B shows the miRFLP assay map, which shows that the absolute fluorescence intensity of the miR-92a and miR-92b fluorescent fragments gradually decreases as the number of molecules to be tested decreases.
  • Figure 5B is a dot-correspondence diagram of the copy number of different miRNAs and their relative fluorescence intensities.
  • the trend is in accordance with the law of power-regression.
  • the calibration curves of the molecular number of miR-92b and miR-92a are: 0.0001 ⁇ 7.4237 and 0.0013 ⁇ 6.4765, respectively.
  • the goodness of fit R squared value is greater than 0.98.
  • Table 3 lists the fluorescence intensity measurements of the three cellular RNAs and the measured fluorescence intensities of miR-92b and miR-92a to relative fluorescence intensity (RFU) values using the fluorescence intensities of the dynamic small RNA standards in the respective reactions.
  • RNU relative fluorescence intensity
  • Table 3 The content of miR-92a and miR-92b in total cellular RNA was determined by the miRFLP method. The total RNA concentration of the cells was determined by a Qubic 2.0 fluorescence meter.
  • the miR-2, miR-92a and miR-92b nucleic acid small molecules which were mixed in equal amounts were diluted to a target of 3 times dilution, and the miRLP analysis conditions of miR-92 in Example 3 were used.
  • the relative fluorescence intensities of the synthetic miR-25, miR-92a and miR-92b at nine different dilutions were determined using the primers and dynamic small RNA molecular standards in Table 5.
  • the number of synthetic miRNA molecules contained in each reaction was: 250,000, 83,333, 27,778, 9,259, 3,086, 1,029, 343, 114, 38 equally mixed miR-25, miR-92a and miR-92b synthetic small RNAs and Blank control.
  • the molecular weight calibration regression curves of miR-92b, miR-92a and miR-25 were: 4.3965 ⁇ 3.4854, 9.9139 ⁇ 3.0082 and 4.4131 ⁇ 3.3164, respectively, and the goodness of fit R squared value was greater than 0.98.
  • Table 4 lists three groups of replicated experiments.
  • the error range (CV) of the relative fluorescence intensity at different molecular level shows that the miRLP analysis of miR-25 has an effective quantitative detection range of 38-250,000, and the detection error range is 104% at the level of 38 molecules. A relatively quantitative determination of the target to be measured outside the detection range is possible.
  • An important reason for miRNAs as ideal biomarkers is that the number of miRNAs varies widely due to physiological pathology and has a high diagnostic sensitivity.
  • the miRFLP analysis shows that the miRNA quantitative determination range, detection error level and objectivity are beyond all current identification methods, satisfying the requirements of clinical miRNA analysis, and has a good application prospect.
  • let-7b, let-7c, let-7d, and let-7g were determined by partial let-7 miRFLP assays using the primers listed in Table 6 and the dynamic miRNA molecular standards. Relative fluorescence intensity. The number of synthetic miRNA molecules added as a template in each reaction was 12,500.
  • let-7c (SEQ ID NO. 14) is: ugagguaguag guuguaugguu
  • let-7d (SEQ ID NO. 15) is: agagguaguag guugcauaguu
  • let-7g SEQ ID NO. 16
  • the sequence of let-7g is: ugagguaguag uuuguacaguu
  • the resulting miRFLP map is shown in Figure 7.
  • the miRFLP assay has high assay specificity and can distinguish miRNA molecules with single base differences.
  • the results confirmed that the maximum cross-reactivity between Let-7 members was in Let-7b and Let- Between 7f and less than 5%, similar to the specificity of ABI's stem-loop primers.
  • the difference is that the cross-reaction in the present invention is determined by using an omega-mixed probe capable of detecting all Let-7 members, that is, This is done in a multi-target assay, rather than in a single primer, and this specificity assessment is closer to the practical application.
  • Table 6 miRFLP quantitative assay for the determination of some of the let-7 family members (let-7b, let-7c, let-7d, let-7g) omega primers (base sequences are SEQ ID NO. 17, SEQ ID NO, respectively) .18, SEQ ID NO. 19, SEQ ID NO. 20, the base sequence of the 3' oligonucleotide-adaptive primer (3'Adapter) used is SEQ ID NO. 21) Dynamic miRNA standard (Std 1 , 2, 3) and composition
  • the relative fluorescence intensities of miR-25 and miR-92b were synthesized according to the miRFLP assay conditions of miR-92 in Example 3 using the stem loop primers and dynamic small RNA molecular standards listed in Table 7.
  • the analysis map correctly displays the DNA fragment representing the target miRNA, and the fluorescence intensity of the fragment is directly proportional to the amount of the target miRNA used, indicating that after some optimization, the stem-loop primer can also be used for the miRFLP analysis of the miRNA.
  • the miRFLP analysis profile in Figure 8 shows the quantitative determination of miR-92b and miR-25 using stem-loop primers. In Figure 8, the correct DNA fragment representing miR-92b is 94.93 nt.
  • the right panel shows the miRFLP profile of miR-25, which represents the correct DNA fragment length of 80.74 nt for miR-25.
  • the relationship between the signal fluorescence intensity measured by the stem-loop primer miRFLP assay and the number of different miRNA templates is similar to that of the omega-gamma miRFLP assay.
  • the stem-loop primer can also be used instead of the omega primer to measure the absolute identity of the miRNA in the same way. Quantitative value.
  • the original design of the stem loop primer was to initiate the reverse transcription reaction of the miRNA.
  • the original intention was to determine the concentration of the target miRNA by using the qPCR amplification cycle number, and quantitatively determine the PCR product with the complementary sequence by using the fluorescently labeled hybridization probe. Therefore, the manner in which the target RNA is identified by the polymorphism of the length of the PCR fragment is not considered.
  • Stem loop primers can be used for reverse transcription of miRNAs, and can also be applied to miRFLP assays as primers for cDNA synthesis.
  • the introduction of different base numbers between the PCR target sites and probes of the traditional stem-loop primers can encode different miRNA targets in the same reaction, satisfying the need for simultaneous detection of multiple miRNAs.
  • Figure 9 illustrates a design of a stem-loop primer probe that is polymorphic for miR-25 and miR-92b, encoded by base number. Length-encoded stem-loop primers can also be combined with different lengths of 3' oligo-adaptive primers to increase the measurable number of miRNAs.
  • the PCR fragment of primer A is 67.17 nt in length
  • the PCR fragment of primer B is 70.08 nt in length, which can be distinguished on the miRFLP analysis spectrum.

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Abstract

本发明提供了一种利用扩增DNA片段长度多态性测定短链RNA的方法，属于分子生物技术领域，包括以下步骤：首先利用至少两种与待测短链RNA相比无天然同源序列的合成小RNA作为测定的内标，将这些合成小RNA以不同分子数混合，形成动态小RNA标准分子梯度；再以等量的所述动态小RNA标准与所述待测短链RNA混合，经由RNA反转录、cDNA加尾、PCR同步扩增及PCR产物DNA长度多态性片段的荧光定量分析，测得所述待测短链RNA扩增产生的DNA片段荧光强度在所述动态小RNA标准荧光强度梯度上的相对比例。

Description

一种利用扩增DNA片段长度多态性定量测定短链RNA的方法 技术领域
本发明涉及分子生物技术领域，特别涉及一种用扩增DNA片段长度多态性定量测定短链RNA的方法。
背景技术
microRNA(即miRNA)是一类调控基因表达的短单链小分子核糖核酸，由22个左右核苷酸(nt)组成。miRNA参与细胞的各种活动，影响并参与维持细胞的生理和分化状态，特定miRNA表达量的变化还能反映生物的生理、病理特征，预示其作为鉴定多种疾病的生物标记的可能性。例如肝细胞所特有的miR-122，在血液中的水平变化就可以揭示肝脏受损的情形。miR-122为肝脏细胞所独有，并在每个人类肝细胞中有13万个，具有目前用于肝损伤鉴定的生物标记物所欠缺的专一性及灵敏度。尽管miRNA在这些病变中所起的作用还有待明确，机制有待进一步的揭示，但这已成为目前miRNA研究的一个最重要的方向。其中miRNA定量检测方法的客观性、灵敏度及标准化是将miRNA研究成果推向临床疾病诊断应用的瓶颈和关键。
已知人类拥有大约1千种以上的miRNA，与各种特定生理病理现象相关的miRNA表达量的动态范围很大，从个位数级别到上万，加上miRNA拥有超短的长度、相似的序列、二级构象、末端修饰及长度差异性等特性，给miRNA的纯化和准确定量带来很大的挑战。
目前应用较多的对miRNA进行定量测定的主流技术包括定量反转录PCR(RT-qPCR)及同时测定多种miRNA的能力的miRNA深度测序法(miR-seq)和microArray杂交测定法等。其它也有很多基于RNA/DNA杂交技术的高通量技术平台，如NanoString nCounter、nanopore技术，但受制于这些方法本身的不完善，缺乏应有的灵敏度和准确性，距离实际应用尚需时日。
上述miRNA测定方法实际应用的主要问题是灵敏度，如其中最灵敏的NanoString nCounter法的测定低限为1amol即约6x105个分子以上的水平，而miR-seq和microArray的测定低限为0.1fmol即6x107个分子以上的水平，显然不满足大多数分子诊断的需求。其中miR-seq法，对miRNA的鉴定不依赖于miRNA序列，可以对miRNA新种的发现有不可替代的作用。而且其能统计出miRNA的相对读数，而有很多研究中利用它作了很多病理诊断应用方面的探索，发现了很多有益的苗头。但是，miR-seq的样品处理需要利用RNA Ligase、DNA Ligase制备适用的cDNA库，并经PCR扩增后才能上机检出，作到相对定量。冗长的样 本制备过程及多种修饰酶的利用，破坏了待测目标数量与检测读值的线性相关性，影响miRNA测定的客观性。因此，如果没有更好的制备方法，miR-seq还不能作为miRNA定量测定的方法。
反转录实时荧光PCR定量技术(RT-qPCR)是现代分子生物诊断方法中对RNA检出最灵敏、最客观可靠的方法，而且方法快捷，不需要特殊的设备。传统的RT-qPCR测定对象是长链的RNA，如mRNA，所用primers长度通常在20nt以上。但是，成熟miRNA的总长度才22nt左右,测定miRNA的RT-qPCR必须经过特殊的修饰，使之能适合qPCR。除茎环引物可作miRNA的RT以外，锁核苷酸引物(LNA)及多聚A聚合酶加尾-寡聚dT引发的RT也被应用于miRNA的定量测定。以利用TaqMan探针、茎环引物的反转录-qPCR方法为代表的实时荧光PCR定量法具备有测定单拷贝miRNA分子的能力，已经广泛的用于miRNA的机制研究、疾病诊断、治疗预后等的研究中。因其是所有方法中最灵敏、客观、容易应用，是验证从microArray、miR-seq方法获得的miRNA表达谱的优选方法。尽管不断的技术改进和对工艺的强化不断地催生新的miRNA qPCR检测方法，但是，至少有两大障碍阻止了这些技术在临床应用中的接受度：方法质量的评估及方法的标准化。这两个障碍严重影响到方法结果在不同批次、操作人员及实验室之间的重复性，即测定结果存在不一致不稳定的缺点。
对液态的生物标本而言，miRNA拷贝数的绝对定量分析可以揭示miRNA在一定体积中的数量，是一种更容易理解和符合自然的单位，尤其适合液体样本。拷贝数的绝对定量分析有助于更准确地理解miRNA与疾病之间的关系，更能精细的揭示病变程度及预后。Bissel等用10倍稀释的合成miRNA等摩尔混合物作模板，利用microArray杂交测定法做出miRNA拷贝数的效正曲线，做到了对miRNA的绝对定量。并实际测定大鼠肝脏RNA中miR-122的含量为每10pg约53000个拷贝。很遗憾的是该法的最低测定的限度为1amol，也即6x105拷贝，不能满足临床诊断的实际需求。实际上，因为不同microArray平台技术的原理均以DNA/RNA杂交来实现的，杂交信号没有扩增，受杂交的热力学平衡的制约，在满足测定的专一性的前提下，方法灵敏度很难突破amol的低限。另外一个可能带来差异的因素是miRNA的标记反应受miRNA的末端序列、二级构象及所用的标记方法所限，很难做到等比例地标记所有种类的miRNA，对不同miRNA的测定带来歧视性偏差。
实用的miRNA定量测定方法必须能作到灵敏、准确和可标准化。临床样品具多样性，及成分的不确定性。一个简单的例子是来自不同个体的血清所含的成分对反转录反应及PCR反应可能没有作用、抑制或促进。现行的miR-seq、RT-qPCR法及microArray法用到RNA ligase、Reverse Transcriptase及Taq polymerase对RNA样品的杂质很敏感，加上这些 技术设计原理是试图直接得到样品中待测目标的数量，而这种测定方式静态地假定纯化RNA为测定对象，没有设计抗衡RNA样品中不纯物影响的机制，使其所得的结果受样品来源影响而呈现随机性，增加了其标准化的难度。因而从严格意义上来说，绝对定量是不可能用现有测定方法来实现的。对于血液等生物样本来讲，从不同个体血清纯化出的RNA中所含的杂质对RT反应及PCR扩增效率具有抑制甚或促进的作用，加大了使用传统RT-qPCR方法进行定量测定的难度。另外样本的处理和纯化，一般采用盐酸胍苯酚等蛋白质变性剂抽提方法得到纯度很高的RNA，但由于miRNA的平均大小仅为22nt，需要高浓度乙醇长时间沉淀以避免丢失，使纯度和产率这对矛盾更加突出，而用亲和树脂柱的纯化方法一般能保证RNA的纯度但尚需证明纯化后RNA的完整性，即miRNA的丢失和选择性丢失的问题。从病人获得的病理标本通常是少量而不可重复的，因此，这些问题一直困扰着将miRNA定量检测技术推向临床应用的努力。
发明内容
本发明的发明目的在于：针对上述存在的问题，提供一种改善短链RNA定量测定的灵敏度、准确度和标准化的新方法。
本发明采用的技术方案是这样的：一种利用扩增DNA片段长度多态性定量测定miRNA的方法，包括以下步骤：
首先利用至少两种与待测短链RNA相比无天然同源序列的合成小RNA作为测定的内标，将这些作为内标的所述合成小RNA以不同分子数混合，形成动态小RNA标准分子梯度；
再以等量的所述动态小RNA标准与所述待测短链RNA混合，经由RNA反转录、cDNA加尾、PCR同步扩增及PCR产物DNA长度多态性片段的荧光定量分析,测得所述待测短链RNA扩增产生的DNA片段荧光强度在所述动态小RNA标准荧光强度梯度上的相对比例，从而实现所述待测短链RNA的相对定量。
在本发明中，首先在样品中加入不同分子数目作为动态小RNA标准的合成小RNA与待测短链RNA混合，同一反应中进行同步检测，由同步测得的分子标准量为动态定量的标准尺度，获得待测短链RNA的相对峰值。为了不与样品中的待测短链RNA的序列相混，所以作为内标的合成小RNA要求与待测短链RNA无天然同源序列。杂质或影响反应的因素对样品中的待测短链RNA及动态小RNA标准分子均有相同程度的影响，这种设计思路明显地区别于现行的方法，使本发明的方法拥有足够的灵敏度和准确性的同时，兼具有对样品不纯成分的一定包容能力，有益于不同操作人员及实验室之间不同样品测定结果的比较，容易实施标准化。从而可以抗 衡RNA尤其是短链RNA纯化、样本质量及仪器、操作、耗材等外在因素对RNA尤其是短链RNA测定结果的影响。
作为优选的技术方案，测得所述待测短链RNA在所述动态小RNA标准梯度上的相对强度比例后，再以待测短链RNA序列为模板合成的短链RNA参照物，该短链RNA参照物的序列与待测短链RNA的序列相同，其不同分子数量，在上述动态小RNA标准为梯度上的相对比例而获得的分子数目-相对强度校正曲线，利用所述待测短链RNA相对强度比例通过所述校正曲线，计算出样品中待测短链RNA的绝对分子数量。
对比用已知数量的合成目标RNA与相同小分子RNA标准的相对测定值作出的校正曲线而得到确定的待测RNA的绝对数目，从而可以获得待测RNA的绝对定量结果。
作为优选的技术方案，所述待测短链RNA为miRNA或siRNA。
本发明的方法可用于对各种生物样本或纯化RNA中miRNA、siRNA及RNA的种类(定性)及含量(定量)的测定，siRNA具有与miRNA一样的物理、化学特性，上述的方法同样适用于siRNA的定性定量测定。本发明也适用于鉴定RNA中的一段或几段较小的序列，从而达到各种不同RNA的定性、定量测定的目的，尤其适合于miRNA这类短链RNA。
越来越多的研究已经揭示miRNA及小RNA表达水平变异对疾病的诊断及预后的联系，从病人获得的病理标本通常是少量而不可重复的，miRNA及小RNA的纯化方法还不能满足现行鉴定方法的需求，本发明可利用抵消miRNA样本中不纯物的影响而对待测miRNA进行精确定量，可以满足通过对miRNA的相对或绝对定量测定达到临床诊断、预后、刑侦等的需求。miRNA指纹谱(miRNA signature)是几个到几十个与某生命现象相关的miRNA在特定样本中的表达水平，其改变规律可揭示相关的生理或病理变化。本发明的实施，可以在一个或几个反应里，方便、快速、灵敏和准确地鉴定miRNA指纹谱，有助于miRNA指纹谱的研究和在临床诊断中的应用。
本发明设计了通过DNA片段长度多态性荧光相对定量分析来完成RNA尤其是miRNA的分子数绝对定量检测的方法(miRNA-derived Fragment Length Polymorphism Assay，本发明中称为miRFLP定量分析法)。miRFLP定量分析法利用3种无天然同源序列的合成小RNA作为测定的内标，将这三种内标小RNA以不同分子数混合，形成动态小RNA标准分子梯度，再以等量的动态小RNA标准与待测短链RNA混合，经由RNA反转录、cDNA加尾、PCR同步扩增及PCR产物的荧光片段长度多态性分析等四个步骤测得待测短链RNA产生的DNA片段荧光强度在动态小RNA标准荧光梯度上的相对比例。用合成的待测短链RNA参照物分子数与其测得的荧光相对强度即可绘出短链RNA绝对数量的换算曲线，由此计算出待测短链RNA的绝对含 量。
作为优选的技术方案，所述RNA反转录过程中，所采用的引物为欧米茄引物。
本发明所述的欧米茄引物，尤其指申请号为PCT/CN2013/070525的PCT专利申请中所公开的欧米茄引物。欧米茄引物作为本发明miRFLP定量分析反应的引物探针。欧米伽引物含有一个极稳定的二级茎环结构，可避免引物二聚体的形成，提高末端引发精确度，且将引物有效地分隔为探针区及编码区。通过加减编码区的碱基数目就可以方便地对不同引物进行长度编码，并可在同一反转录反应中将多种miRNA转换为长度不一的cDNA片段,经PCR同步扩增进行荧光片段长度多态性分析，解决了单一反应中检测多种miRNA的关键技术问题。
miRFLP反应由miRNA反转录、cDNA加尾、PCR同步扩增及PCR产物的荧光片段长度多态性分析等四个步骤组成，见图1。反应的第一步是将miRNA和欧米伽引物混合杂交，miRNA与互补的探针形成配对，然后用反转录酶以未配对的miRNA 3’末端为模板合成cDNA。除去RNA后，新合成的cDNA与含有共同PCR靶位的3’寡居核苷酸引物杂交并用DNA聚合酶以cDNA的3’末端为起始补齐DNA配对后的单链缺口。经此组合后，正确装配的cDNA拥有相同的5’及3’末端序列，可经一对荧光PCR引物进行同步等比扩增，扩增产物通过毛细管凝胶电泳分离后测定不同长度PCR片段的荧光强度，完成miRFLP分析图谱。
作为优选的技术方案，所述RNA反转录过程中，所采用的引物为茎环引物。
作为进一步优选的技术方案，所述茎环引物为经过长度编码的茎环引物。
作为更进一步优选的技术方案，所述长度编码的方法为：在所述茎环引物的PCR靶位点与探针序列之间加上不同碱基数目，并调整引物5’端的碱基序列使之维持茎环的二级结构不变。
茎环结构的引物探针(Applied Biosystems,Inc,如PCT/CN2013/070525所提及的)将多个小RNA同时定量地转换为cDNA，经与上述miRFLP反应路径相似的cDNA产物加尾、反应后经PCR同步扩增进行荧光片段长度多态性分析。实验结果证实，只要加上长度可变的编码区，茎环引物也可用于本发明的设计规划的实施，并在测定有限数目的miRNA目标时，可以与欧米伽引物互换或同时使用。
作为优选的技术方案，所述校正曲线符合对数回归的方式，表述为：aXb，其中a、b为常数项，经对不同分子数目的所述合成小RNA的实际测定值而确定。
作为优选的技术方案，所述与待测短链RNA相比无天然同源序列的合成小RNA的种数为三种。由于荧光强度和荧光物质的数量不是线性的关系，需要至少3种来确定一元二次方程的回归，而采用三种合成小RNA，既可以达到定量测定的要求，同时检测过程也较经济。
miRFLP定量分析法中的每一步中间产物的实现和信号的扩增都是以线性或接近线性定量的方式进行的，因此扩增后DNA片段的荧光强度与目标miRNA的分子数成线性定量的关系。但受DNA分析仪荧光探头的线性定量范围的客观限制，仪器探头的荧光强度与荧光物质的数量间的响应曲线适用于实测得到的待测目标的荧光强度与其分子数之间的换算。为确定仪器探头的响应曲线，我们选择了一份具有多个杂峰片段的PCR产物，经系列稀释后将样品用ABI3730xl型DNA分析仪进行荧光定量分析，分析确定了稀释倍数与实测的荧光强度的相关性在25–25000FU的测定范围内均符合一元二次方程的回归关系，R2平均值大于0.9999。本方法中所用的动态miRNA标准和样本目标荧光信号的扩增是以接近线性的定量方式同步进行的，一元二次回归方程适用于待测miRNA对动态miRNA标准的相对荧光强度的计算。现代的DNA测序仪具有极高的敏感度和动态探测范围(ABI 3730DNA分析仪：5-31000FU)。不同厂家或不同型号的DNA荧光定量分析仪均可采用上述方法对适用的荧光响应曲线及范围进行标定，遴选出最适合亦即最精确的回归方式。
由于各种外在因素对测定方法的影响，所测得的miRNA值还不是miRNA的绝对分子数目，而是待测miRNA在动态miRNA标准的荧光强度梯度上的比值，比值的大小反映出待测miRNA分子数目与动态miRNA标准分子数的比例。因待测miRNA与动态miRNA标准在同一反应中检测，同步扩增，排除了外在因素的干扰，动态miRNA标准的分子数目用量是恒定的，待测miRNA相对荧光强度比值就能客观地反映不同反应中待测miRNA的多寡。利用系列稀释的已知分子数的合成待测miRNA为参照物与动态miRNA标准的比较可以作出待测miRNA的相对比值与待测miRNA分子数目的校准曲线，将待测miRNA相对比值转换为其miRNA绝对拷贝数。用二倍或三倍稀释的合成miRNA参照物还可以用来准确地衡量方法的测定范围及在每个浓度点的大致误差范围，可以帮助提高所得的数据分析结果的可信度和标准化。用IBM SPSS Statistics 20分析软件对10个实际测得的三倍系列稀释的数据进行回归模型的分析，我们发现miRNA的相对荧光强度与分子数之间符合对数方程的回归规律(power regresssion)，拟合优度R2在适用的测定范围内大于0.99。校正miRNA参照物易于标准化，可用于校正不同操作员之间的操作误差，让不同实验室得出的数据具有可比性。
由于不同miRNA的探针及3’适配寡聚核苷酸具有不同的Tm值，即使在相同的反应条件下，其热动力学的影响也导致不同miRNA拥有不同的校准曲线。由于没有能通用的miRNA的绝对定量校准曲线，每个待测miRNA均需有自己的绝对定量标准曲线。本发明适用于多个miRNA的同时鉴定，多种miRNA合成参照物也可以混合使用，大大减少所需反应的数目。例如完成9个不同miRNA的校正曲线，以三倍稀释制作一个覆盖50-5x105个分子测定范围的标准曲线需要测 定10个不同浓度的标准miRNA参照物，如果使用标准的RT-qPCR测定法，仅完成标准曲线的制作就需要至少90个反应才行，而用本发明的多目标同步测定法可将9种合成miRNA混合后作为模板，9种miRNA的绝对定量校准曲线仅需10个反应就可完成。
综上所述，由于采用了上述技术方案，本发明的有益效果是：本发明的miRFLP定量分析法与现行方法相比，至少有如下优点：
1.绝对定量：得益于与样本RNA在同一反应管中反应形成的动态小RNA标准分子，消除复杂样品以及RNA纯度对测定的干扰，可对待测miRNA进行绝对定量；
2.高特异性：可鉴别只有1个碱基差别或同一家族的miRNA；
3.高灵敏度：对细胞单拷贝miRNA测定的上样量范围为0.1μg-1ng总RNA；
4.测定范围：在100-106拷贝数范围内准确定量，超出此范围可作相对定量；
5.结果客观：设立了内控做为RT或PCR反应失败导致阴性结果的指标，对基因片段的甑别可以精确到±0.2个碱基；
6.重复性好：可控的合成miRNA标准参照保证操作人员及实验室之间的重复性和可比性，有益于方法的标准化。
7.适用于RNA粗提物：可直接从血清、血浆等病人样本中准确客观地测出miRNA的表达水平，重复好，结果可靠。
附图说明
图1A和图1B是miRFLP定量分析法反应流程及测定原理图；
图2是随机选取的具有多个杂峰的荧光PCR产物经25倍稀释后的DNA片段长度荧光分析图谱；
图2中，纵坐标FU为荧光强度，“□”标注的为随机选择的DNA片段；
图3是长度为62.00nt及91.22nt(B)的DNA片段在不同稀释度下的荧光测定值与稀释倍数关系符合一元二次方程回归曲线；
图3中，“A”为62.00nt的DNA片段，“B”为91.22nt的DNA片段，纵坐标FU为荧光强度单位；
图4A和图4B是动态miRNA标准和不同浓度miRNA的miRFLP分析图谱；
图4A和图4B中，“std 1”、“std 2”及“std 3”分别代表动态miRNA标准(std 1、std 2和std 3)的荧光DNA片段，“1”、“2”、“3”、“4”分别代表反应1、反应2、反应3、反应4，“a”代表miR-92a的荧光DNA片段，“b”代表miR-92b的荧光DNA片段，纵坐 标为荧光强度；
图5A和图5B是不同分子数miR-92a和miR-92b的miRFLP测定图谱及标准曲线；
图5A和图5B中，“1”为只含有动态miRNA标准的空白对照测定结果，“2-10”分别为加入了2.5x105个至244个合成miR-92a分子和等量的miR-92b为待测目标，图5B为不同miRNA拷贝数与其相对荧光强度的点对应图；
图6是miRFLP方法测定miR-25家族成员矫正曲线的重复性及测定范围结果图；
图6中，横坐标为miRNA分子数，纵坐标为相对荧光强度；
图7是Let-7家族miRFLP定量分析谱对单个Let-7成员的测定结果图；
图7中，从上至下分别为let-7b、let-7c、let-7d及let-7g的测定结果，“std 1”、“std 2”及“std 3”代表的含义与图4A中的“std 1”、“std 2”及“std 3”一致；
图8是用茎环引物的miRFLP分析方法对miR-92b及miR-25的测定结果；
图9带有以碱基数目为编码的茎环引物；
图9中，A：为探测Standard 1RNA设计的茎环引物，在5’端PCR靶位(箭头)与探针之间放置了4个碱基(方框)作为标记。茎环dG＝-13.71。
B：为探测Standard 2RNA设计的茎环引物，在5’端PCR靶位(箭头)与探针之间放置了7个碱基(方框)作为标记。茎环dG＝-13.09。
具体实施方式
下面对本发明作详细的说明。
为了使本发明的目的、技术方案及优点更加清楚明白，以下结合实施例，对本发明进行进一步详细说明。应当理解，此处所描述的具体实施例仅仅用以解释本发明，并不用于限定本发明。
实施例1 miRFLP定量分析法反应流程及测定原理
本实施例以欧米伽引物为例，具体阐述miRFLP定量分析法反应流程及测定原理。
miRFLP反应由miRNA反转录、cDNA加尾、PCR同步扩增及PCR产物的荧光片段长度多态性分析等四个步骤组成，如图1A和图1B所示。反应的第一步是将miRNA和欧米伽引物混合杂交，miRNA与互补的探针形成配对，然后用反转录酶以未配对的miRNA3’末端为模板合成cDNA。除去RNA后，新合成的cDNA与含有共同PCR靶位的3’寡居核苷酸引物杂交并用DNA聚合酶以cDNA的3’末端为起始补齐DNA配对后的单链缺口。经此组合后，正确装配的cDNA拥有相同的5’及3’末端序列，可经一对荧光PCR引物进行同步等比扩增，扩增产物通过毛细管凝胶电 泳分离后测定不同长度PCR片段的荧光强度，完成miRFLP分析图谱，图1B为整个miRFLP定量分析的最后一步。
实施例2 PCR荧光标记产物等倍稀释液荧光强度的测定试验
荧光定量分析仪测出的荧光强度与待测荧光物质的数量成正比关系，这种关系与仪器所配置的荧光探头有关，即不同的荧光探头拥有不同的荧光响应曲线。ABI的Prizma 310型DNA测序仪测得的荧光强度在5-7000FU的范围里与待测的荧光物质数量成线性定量的关系，当荧光强度值大于7000FU时改变为抛物线。不同仪器的荧光探头响应特性不一样，可以影响到测出的荧光强度与待测荧光量的回归关系。本实施例对ABI 3730xl型DNA分析仪的荧光响应曲线用2倍系列稀释的PCR产物进行了标定。实验随机选取了一个具有多个不同片度且荧光强度变化较大的荧光PCR产物，用1xTE以25、50、100、200及400倍稀释后用ABI 3730xl型DNA分析仪分析。图2是该PCR产物经25倍稀释的样品荧光扫描图，从中选取11个峰值介于253-25000FU的DNA片段，统计出它们在不同稀释度的荧光读值(见表1)，经IBM SPSS Statistics 20统计软件对稀释倍数和荧光强度测定值的对应关系进行回归曲线的优化评估，显示一元二次方程回归曲线适用于描述所有片度的荧光强度与稀释倍数的关系，回归的拟合优度R平方值均大于0.999。因此，经ABI 3730xl型DNA分析仪测得的DNA片段的荧光强度与荧光物质数量的对应关系可以用一元二次方程回归曲线来精确计算，而不是简单的线性定量关系。重复实验证明同样的样品在不同批次的测定绝对值可以有所不同，但这种一元二次方程的回归模型并不受影响。图3显示了在低丰度片段(A：30FU-312FU)及高丰度片段(B：2052FU-24949FU)经系列稀释后的回归曲线图。不同厂家或不同型号的DNA荧光定量分析仪均可采用上述方法对适用的荧光响应曲线及范围进行标定，遴选出最适合亦即最精确的回归方式。
表1：用于图2的PCR产物经25、50、100、200和400倍稀释后用ABI 3730xl型DNA分析仪在不同稀释倍数下测得的荧光值及经IBM SPSS Statistics 20统计软件作一元二次方程回归统计后的拟合优度R平方：
Figure PCTCN2015089566-appb-000001
实施例3 miR-92a和miR-92b的miRFLP定量分析测定试验
miRFLP分析法将待测miRNA与动态miRNA标准混合均匀，经过miRNA反转录、cDNA加尾修饰及荧光PCR同步扩增处理，最后用DNA测序仪进行DNA片段长度和荧光定量的分析。具体做法，首先配制4μl杂交母液：2μl 5xRT缓冲液、1μl 10mM MgSO4、1μl动态miRNA标准的混合物，该混合物包括分子数为3×106的“标准1RNA”(即图4A中的“std1”，下同)、分子数为×105的“标准2RNA”(即图4A中的“std2”，下同)、分子数为3×104的“标准3RNA”(即图4A中的“std3”，下同)，动态miRNA标准的RNA序列见表2，经Sangers miRbase20版数据库所做的序列搜索证明无同源序列)。用4μl杂交母液同2μl待测RNA样本混合，加入2μl 10nM的欧米伽探针混合物(含：针对std1，2，3及miR-92a和miR-92b的探针，探针序列见表2)。混合均匀后进行杂交，杂交反应条件为：55C 10分钟，然后以每分钟一度的速度从55C降到20C。加入2μl反转录酶混合液(1μl MMTV反转录酶，0.5μl dNTP，0.5μl水，Takara)，混匀后25C保温30分钟，37C10分钟，85C变性5分钟。取5μl RT产物加入15μl 3’适配引物缓冲液(10μl JumpStartTM Taq ReadyMix，2μl 100nM 3’寡聚核苷酸适配引物,1μl 0.1ug/μl RNase A，2μl无菌水，Sigma)，反应条件：95C 2分钟，60c 10分钟，重复5次55c 1分钟到30c 5分钟的循环，20c保温30分钟,以每分钟1C的速度从42c升到68c,72c保温5分钟。取5μl加入由15μl JumpStartTM Taq ReadyMix(Sigma)，0.5uM PCR引物和10μl水配成25μl的PCR反应液。进行荧光PCR扩增，反应条件为：95℃ 2分钟，40次经95℃ 10秒，68℃ 3分钟，72℃ 30秒的循环。通用PCR引物为：[5Fam]GTGCTGAGTCACGAGGTATTCTA及CACCGACAGGAGACCTGTTCT(购自GenScript)。反应完成后，将PCR产物以1:20或1:50的稀 释度稀释后用ABI 3730xl型DNA分析仪进行荧光片段长度多态性分析。miRFLP分析图谱结果见图4A和图4B所示；
图4A中，反应1为只含有动态miRNA分子标准的空白对照测定结果；反应2中加入了2.5x105个miR-92a分子和等量的miR-92b为待测目标，图4B中，反应3中加入了3.1x104个miR-92a和等量的miR-92b为待测目标，反应4中加入了78pg H1299细胞总RNA。
在图4A和图4B的miRFLP分析图谱中，不同长度的DNA片段代表不同的miRNA分子(峰注上面的数值为片段长度，nt)，其荧光强度反映出该miRNA的相对数量(峰注下面的数值为荧光强度，FU)，同一反应中不同动态miRNA标准测得的荧光强度形成与其分子数相应的荧光强度梯度。不同反应中按比例放入的各种动态miRNA标准的绝对数目是一致的，但因诸多原因，如操作、仪器、耗材、试剂、测定条件等的不同质性，均影响到荧光强度的绝对测定值，并显示出不小的差异(如反应1和反应3中的std 1的峰值)。而因待测目标的稀释倍数较小，反应2中用于RNA保存的保存液浓度要高于反应3，影响到RT或PCR反应的效率，引起动态miRNA标准荧光强度梯度的变动。因待测目标与动态miRNA标准反应前已经混匀，这些影响反应效率的因素对待测目标和动态miRNA标准的影响是相同的，待测目标产生的荧光强度在动态标准荧光强度梯度的相对比例可以用来客观地衡量待测目标与动态miRNA标准之间的分子数比。
表2：miRFLP定量分析法测定miR-92a及miR-92b的欧米伽引物(其碱基序列分别为SEQ ID NO.1、SEQ ID NO.2；采用的欧米欧米伽引物的碱基序列均为SEQ ID NO.3；采用的3’寡聚核苷酸适配引物(即表2中的“3’Adapter”)的碱基序列分别为SEQ ID NO.4、SEQ ID NO.5)、动态miRNA标准(Std 1，2，3)(其碱基序列分别为SEQ ID NO.6、SEQ ID NO.7、SEQ ID NO.8；分别采用的欧米欧米伽引物的碱基序列分别为SEQ ID NO.9、SEQ ID NO.10、SEQ ID NO.11，采用的3’寡聚核苷酸适配引物的碱基序列均为SEQ ID NO.12)及成分构成：表中：dG,dC,dA and dT的平均分子量(308.95道尔顿)计为1nt，5'荧光基团分子量:474.5。
Figure PCTCN2015089566-appb-000002
实施例4 miRFLP定量分析校准曲线及细胞总RNA中miR-92a和miR-92b的定量测定试验
miRFLP定量分析反应对同一反应里的小RNA的反转录、修饰及扩增反应均以线性等比的方式进行，动态标准荧光强度梯度可以排除外在因素对测定的影响，但探针的Tm、PCR片段长度及构象的差异仍然能影响DNA片段的荧光强度。通过设计和反应条件的优化可以把后二者的影响降低到可以忽略的程度，但探针Tm对待测目标产生的荧光强度的影响不可忽视。因而需要用已知数目的待测目标作出分子数目与相对荧光强度之间的校准曲线才能将待测目标的相对荧光强度转换为待测目标的实际分子数。用系列稀释的合成miRNA作为待测目标，可获得相对荧光强度与目标分子数的转换曲线。
本实施例利用等量混合的合成miR-92a及miR-92b核酸小分子以2倍的稀释度稀释后为待测目标，遵循实施例3中的miR-92的miRFLP分析法的测定条件，测定了9个不同稀释度的合成miR-92a和miR-92b的相对荧光强度。实验组设置了对3.12ng、0.312ng、0.0312ng A549细胞RNA，0.222ng Hela细胞RNA及0.25ng H1299细胞RNA的测定反应。每个测定反应均设置三次重复实验，以判定测定的误差范围。图5A中左边的数字显示每个反应中miR-92a和miR-92b的分子数目。右边为miRFLP测定图谱，显示miR-92a及miR-92b荧光片段的绝对荧光强度随待测分子数的降低而逐渐下降。用Microsoft Excel表格对动态miRNA标准的荧光强度和对应的分子数作回归统计，求得最佳的一元二次方程，并在此方程中代入待测目标荧光强度即得出待测目标相对于动态小RNA标准的的相对荧光强度。图5B为不同miRNA拷贝数与其相对荧光强度的点对应图，其趋势符合乘方回归的规律，miR-92b及miR-92a的分子数校准回归曲线分别为：0.0001×^7.4237和0.0013×^6.4765，拟合优度R平方值大于0.98。表3列出了三种细胞RNA的荧光强度测定结果以及利用各自反应中动态小RNA标准的荧光强度将测得的miR-92b及miR-92a的荧光强度转换成相对荧光强度(RFU)值。分别利用上述miR-92a、miR-92b的分子校准曲线的乘方回归方程，可以得出miRNA在细胞RNA中的含量及各自的误差范围。对3个10倍稀释的A549细胞RNA的测定结果显示在0.03ng-3ng的RNA上样范围内，miR-92b及miR-92a的测定误差分别为10.13％和12.63％，显示出miRFLP分析法对miRNA的定量测定受样品上样量的影响很小，可靠性大。
表3 利用miRFLP方法测定细胞总RNA中miR-92a及miR-92b含量。细胞总RNA浓度由Qubic 2.0荧光定量仪测定。
Figure PCTCN2015089566-appb-000003
实施例5 miRFLP定量分析法的定量测定范围及误差验证试验
利用等量混合的合成miR-2、miR-92a及miR-92b核酸小分子以3倍的稀释度稀释后为待测目标，遵循实施例3中的miR-92的miRFLP分析测定条件，利用列于表5中的引物及动态小RNA分子标准测定9个不同稀释度的合成miR-25、miR-92a和miR-92b的相对荧光强度。每个反应中所含合成miRNA分子数分别为：250,000、83,333、27,778、9,259、3,086、1,029、343、114、38个等量混合的miR-25、miR-92a和miR-92b合成小RNA以及空白对照。每个测定反应均设置三次重复实验，以判定测定的误差范围。用Microsoft Excel表格对每个反应中动态小RNA标准的荧光强度和对应的分子数作回归统计，求得最佳的一元二次方程，并在此方程中代入待测目标荧光强度即得出待测目标相对于动态小RNA标准的的相对荧光强度。图6显示不同miRNA分子数与其测定的相对荧光强度的散点对应图及对数回归的拟合优度。miR-92b、miR-92a及miR-25的分子数校准回归曲线分别为：4.3965×^3.4854、9.9139×^3.0082及4.4131×^3.3164，拟合优度R平方值大于0.98。表4列出了三组重复实验所 得不同分子数水平的相对荧光强度的误差范围(C.V)显示出miR-25的miRFLP分析法的有效定量检测范围为38-250,000个，在38个分子的水平上的检测误差范围为104％，并可在此检测范围外对待测目标作出相对定量的测定。miRNA作为理想的生物标记物的一个重要原因是由于受生理病理的影响，miRNA数量的变动范围很大，具有很高的诊断灵敏性。miRFLP分析法显示出的miRNA定量测定范围、检测误差水平及客观性，均超出目前所有的鉴定方法，满足临床对miRNA分析的要求，具有很好的应用前景。
表4 miRFLP分析法测定miR-25家族各个成员在不同分子数水平的误差范围
Ref miRNA miR-92b miR-92a miR-25
分子数 C.V C.V C.V
250,000 29.19％ 3.40％ 4.84％
83,333 21.10％ 8.39％ 8.46％
27,778 14.96％ 4.00％ 6.66％
9,259 16.19％ 5.49％ 2.93％
3,086 13.13％ 25.08％ 17.95％
1,029 17.04％ 26.68％ 24.54％
343 13.38％ 17.85％ 23.72％
114 54.04％ 26.95％ 33.07％
38 103.62％ 88.01％  
表5 miRFLP分析法测定miR-25家族成员的欧米伽引物(Omega Primer)、动态miRNA标准(Std 1，2，3)及3’寡聚核苷酸适配引物(3’Adapter)的碱基序列及成分构成表
Figure PCTCN2015089566-appb-000004
实施例6 miRFLP定量分析法的方法专一性验证试验
遵循实施例3中的miRFLP分析测定条件，利用列于表6中的引物及动态miRNA分子标准测定的部分let-7 miRFLP分析谱分别测定let-7b、let-7c、let-7d及let-7g的相对荧光强度。每个反应中所加入的作为模板的合成miRNA分子数量均为12500个。
其中，let-7b的序列(SEQ ID NO.13)为：ugagguaguag guugugugguu
let-7c的序列(SEQ ID NO.14)为：ugagguaguag guuguaugguu
let-7d的序列(SEQ ID NO.15)为：agagguaguag guugcauaguu
let-7g的序列(SEQ ID NO.16)为：ugagguaguag uuuguacaguu
所得的miRFLP图谱见图7。从图7中可以看出，miRFLP测定法具有很高的测定专一性，可以区分单个碱基差异的miRNA分子。用12500个Let-7不同成员作模板，用Let-7家族miRFLP测定谱对单个Let-7成员分别进行独立的测试，结果证实了Let-7成员间的最大交叉反应在Let-7b和Let-7f之间，并小于5％，与ABI公司的茎环引物的专一性相似。但不同的是，本发明中交叉反应的测定是用含有能探测所有Let-7成员的欧米伽混合探针，也即 在多目标测定的反应中实现的，而非在单一引物中进行，这种对专一性的评估方式更贴近实际应用。
表6：miRFLP定量分析法测定部分let-7家族成员(let-7b、let-7c、let-7d、let-7g)的欧米伽引物(碱基序列分别为SEQ ID NO.17、SEQ ID NO.18、SEQ ID NO.19、SEQ ID NO.20，采用的3’寡聚核苷酸适配引物(3’Adapter)的碱基序列均为SEQ ID NO.21)动态miRNA标准(Std 1，2，3)及成分构成
Figure PCTCN2015089566-appb-000005
实施例7 利用茎环引物的miRFLP定量分析测定方法
利用表7列出的茎环引物及动态小RNA分子标准，按照实施例3中的miR-92的miRFLP分析测定条件，测合成miR-25和miR-92b的相对荧光强度。分析图谱正确地显示出代表目标miRNA的DNA片段，并且片段的荧光强度与目标miRNA的使用量成正比例，说明经过一定的优化，茎环引物也可用于miRNA的miRFLP分析法。图8中的miRFLP分析谱显示利用茎环引物对miR-92b及miR-25的定量测定结果。图8中，代表miR-92b的正确DNA片段为94.93nt。右图为miR-25的miRFLP分析谱，代表miR-25的正确DNA片段长度为80.74nt。茎环引物miRFLP分析法测出的信号荧光强度与不同miRNA模板的数量之间关系与欧米伽引物miRFLP分析法结果相似，茎环引物也可用来代替欧米伽引物以同样的方式测出miRNA的绝对定量值。
茎环引物的原始设计用途是用来启动miRNA的反转录反应，初衷是利用qPCR扩增循环数测定目标miRNA的浓度，用荧光标记的杂交探针对具有互补序列的PCR产物进行定量测定。因此并没有考虑利用PCR片段长度的多态性对目标RNA的鉴定的方式。茎环引物可以用于miRNA的反转录，也可应用于miRFLP分析法作为cDNA合成的引物。在传统茎环引物的PCR靶位点和探针之间引入不同碱基数目为编码，可以在同一反应中区分不同的miRNA目标，满足对多个miRNA的同时检测的需要。图9例举了利用碱基数编码的、针对miR-25和miR-92b的长度多态的茎环引物探针的设计方案。长度编码的茎环引物也可与不同长度的3’寡聚适配引物组合，增加miRNA的可测数目。图9中，引物A的PCR片段长度为67.17nt,引物B的PCR片段长度为70.08nt，可以在miRFLP分析谱上区分开来。
表7 miRFLP分析法测定miR-25及miR-92b的茎环引物、动态miRNA标准(Std 1，2，3)及3’寡聚核苷酸适配引物(3’Adapter)的碱基序列及成分构成
Figure PCTCN2015089566-appb-000006

Claims (9)

  1. 一种利用扩增DNA片段长度多态性定量测定短链RNA的方法，其特征在于，包括以下步骤：
    首先利用至少两种与待测短链RNA相比无天然同源序列的合成小RNA作为测定的内标，将这些作为内标的所述合成小RNA以不同分子数混合，形成动态小RNA标准分子梯度；
    再以等量的所述动态小RNA标准与所述待测短链RNA混合，经由RNA反转录、cDNA加尾、PCR同步扩增及PCR产物DNA长度多态性片段的荧光定量分析,测得所述待测短链RNA扩增产生的DNA片段荧光强度在所述动态小RNA标准荧光强度梯度上的相对比例，从而实现所述待测短链RNA的相对定量。
  2. 如权利要求1所述的一种利用扩增DNA片段长度多态性定量测定miRNA的方法，其特征在于，测得所述待测短链RNA在所述动态小RNA标准梯度上的相对强度比例后，以待测短链RNA序列为模板合成短链RNA参照物，测定不同分子数量的待测RNA参照物在上述动态小RNA标准梯度上的相对比例，由此获得待测短链RNA参照物的分子数目-相对强度的校正曲线，再利用所述待测短链RNA相对强度比例通过所述校正曲线，计算出样品中待测短链RNA的绝对分子数量。
  3. 如权利要求1或2所述的一种利用扩增DNA片段长度多态性定量测定短链RNA的方法，其特征在于，所述待测短链RNA为miRNA或siRNA。
  4. 如权利要求1或2所述的一种利用扩增DNA片段长度多态性定量测定短链RNA的方法，其特征在于，所述RNA反转录过程中，所采用的引物为欧米茄引物。
  5. 如权利要求1或2所述的一种利用扩增DNA片段长度多态性定量测定短链RNA的方法，其特征在于，所述RNA反转录过程中，所采用的引物为茎环引物。
  6. 如权利要求5所述的一种利用扩增DNA片段长度多态性定量测定短链RNA的方法，其特征在于，所述茎环引物为经过长度编码的茎环引物。
  7. 如权利要求6所述的一种利用扩增DNA片段长度多态性定量测定短链RNA的方法，其特征在于，所述长度编码的方法为：在所述茎环引物的PCR靶位点与探针序列之间加上不同碱基数目，并调整引物5’端的碱基序列使之维持茎环的二级结构不变。
  8. 如权利要求2所述的一种利用扩增DNA片段长度多态性定量测定短链RNA 的方法，其特征在于，所述校正曲线符合对数回归的方式，表述为：aXb，其中a、b为常数项，经对不同分子数目的所述合成小RNA的实际测定值而确定。
  9. 如权利要求1所述的一种利用扩增DNA片段长度多态性定量测定短链RNA的方法，其特征在于，所述与待测短链RNA相比无天然同源序列的合成小RNA的种数为三种。
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