CN107190091B

CN107190091B - Real-time quantitative PCR method suitable for gene expression quantification of FFPE sample

Info

Publication number: CN107190091B
Application number: CN201710600770.3A
Authority: CN
Inventors: 赵金银; 庞震国; 白山; 刘琦; 李�杰; 许立志; 于闯
Original assignee: Dalian Gentalker Biotechnology Co ltd
Current assignee: Dalian Jingtai Medical Laboratory Co ltd
Priority date: 2017-07-21
Filing date: 2017-07-21
Publication date: 2021-04-20
Anticipated expiration: 2037-07-21
Also published as: CN107190091A

Abstract

The present invention relates to a method for nucleic acid molecule quantification, in particular, to a real-time quantitative PCR method suitable for gene expression quantification of paraffin tissue section (FFPE) samples, comprising 1) a reverse transcription reaction stage; 2) a pre-amplification reaction stage; and 3) a nested in-amplification qPCR reaction stage. Compared with the traditional reverse transcription qPCR, the method not only ensures the specificity of the reverse transcription and qPCR reaction, but also reduces the concentration of the substrate required by the reaction, can improve the detection sensitivity of the qPCR by more than 500 times, and is particularly suitable for the quantitative detection of the mRNA extracted from the paraffin tissue section with low quality and/or low concentration. The method of the invention can greatly improve the sensitivity and reliability of such detection.

Description

Real-time quantitative PCR method suitable for gene expression quantification of FFPE sample

Technical Field

The present invention relates to a method for quantifying nucleic acid molecules, and more particularly, to a real-time quantitative PCR method suitable for quantifying gene expression in paraffin tissue sections (FFPE) samples.

Background

Quantification of gene expression regulation levels is frequently used in medical diagnostics and in physiological, biological pathway function studies. The change of the expression level of a specific gene can be used as a molecular signal for some disease diagnosis, and some disease prognosis diagnosis methods, such as breast cancer remote recurrence prediction diagnosis (Oncotype DX), determine the risk of the breast cancer postoperative remote recurrence according to the expression levels of 16 cancer-related genes and 5 housekeeping genes. The specimen subject examined for the diagnostic protocol was a formalin-fixed, paraffin-embedded tissue section. Formalin-fixed paraffin-sectioned tissue specimens are the most common samples for clinical pathological analysis, but their mRNA integrity is destroyed during preparation. In addition, the poly A structure is also degraded in a large amount, resulting in low efficiency of reverse transcription reaction and even reaction failure. Moreover, since the paraffin sections often contain only a small amount of cells of interest (e.g., cancer cells), it is difficult to perform a diagnostic method based on the detection of gene expression levels in such samples. Currently, the most common technical platform for detecting gene expression level is reverse transcription real-time quantitative PCR (RT-qPCR) method, such as distal recurrence prognosis diagnosis of Oncotype DX breast cancer and the like. qPCR quantification of gene expression levels the most widely used method is relative quantification, measuring the relative expression difference between the target gene and the reference gene at the gene level (delta CT method); the reverse transcription real-time quantitative PCR method for measuring the difference of gene relative expression between different samples on a biological level (delta-delta CT method) is divided into a two-step method and a one-step method experimentally. Two-step method RT-qPCR separates the reverse transcription reaction from the qPCR reaction. The first step of reverse transcription reaction usually uses multiple T primer (polyT) or 6 base random short segment as universal reverse transcription primer to proceed the transcription reaction. The product of the reverse transcription reaction is diluted (or stock solution) and then subjected to a second qPCR reaction using gene-specific primer amplification. The advantage of the two-step RT-PCR is that the reaction system is simple, and the universal primer is adopted to carry out the simultaneous reverse transcription of multiple genes, thus avoiding the disadvantage of the reverse transcription efficiency preference of different reverse transcription primers. However, there are also significant disadvantages, such as low reverse transcription efficiency caused by low effective concentration of the reverse transcription primer of the universal primer for a certain gene, or failure of the reverse transcription primer when the sample is degraded seriously, etc.

In the traditional one-step RT-qPCR, a reverse transcription reaction and a PCR reaction are placed in a reaction system, wherein a 3' end primer of the PCR is simultaneously used as a gene-specific reverse transcription primer and a qPCR primer. The reverse transcription and the PCR reaction are carried out in sequence in the same reaction system, and the traditional one-step method qPCR has the advantage that a gene specific primer is adopted as a reverse transcription primer, so that the concentration of the reverse transcription primer aiming at a target gene is greatly improved. However, the disadvantage is also very obvious, that is, the reverse transcription primer and the PCR reaction primer are the same fragment, the annealing temperature is set as the annealing temperature of PCR, which is much higher than the reverse transcription temperature endured by reverse transcriptase, under the condition far lower than the standard annealing temperature of the primer, a great deal of non-specific reverse transcription can be caused, and the non-specific reverse transcription product can also cause interference in the next PCR reaction because the non-specific reverse transcription product contains the PCR primer matching sequence. In addition, because the design of primers is specific to PCR reaction, the primer concentration and annealing temperature are not optimized for reverse transcription reaction. The gene-specific reverse transcription primers can show different reverse transcription efficiencies under the condition of uniform reverse transcription, which can also cause large systematic errors to the quantitative expression of different genes by the next qPCR, and cause inaccuracy of detection and diagnosis. In order to improve sensitivity and accuracy of RT-qPCR quantification of low-quality or/and small amounts of mRNA, such as formalin-fixed paraffin-embedded tissue sections, there have been some works such as improving RNA extraction quality and improving reagents (using reverse transcriptase with higher temperature tolerance), etc., which have a limited contribution to the objective and do not meet practical objectives and requirements.

In order to improve the sensitivity and stability of RT-qPCR, a more effective strategy is to improve the efficiency of reverse transcription and PCR amplification. One of the current strategies aimed at increasing the efficiency of reverse transcription of RNA, especially for very small and/or low quality RNA, is to introduce a linker sequence at one or both ends during reverse transcription, pre-amplify with linker primers and then perform nested amplification with gene-specific primers, such as Clontech's cDNA synthesis kit (see

PCR cDNA Synthesis Kit) greatly improves the sensitivity of target gene segment amplification. However, this strategy is only suitable for PCR of qualitative nature, which aims to obtain a sufficient amount of the target fragment and cannot effectively limit the amplification of non-specific products, and the gel cutting recovery is a necessary step for obtaining specific amplification products. This strategy is therefore not practical for real-time quantitative PCR. But the basic strategy provides a specific strategy for improving the reaction efficiency of RT-qPCRAnd (6) a thought.

As another example, to improve the sensitivity of real-time quantitative PCR, there have been patents and literature relating to the introduction of nested reactions into quantitative PCR (e.g., PCR Methods applied. 1994 Jun; 3(6): 332-7; Journal of microbiological Methods 102(2014):15-22) using two pairs of primers specific to genes to improve the sensitivity of real-time quantitative PCR. In order to ensure the reaction efficiency and specificity of the nested reaction, the annealing temperature and concentration ratio of the inner primer and the outer primer of the nested quantitative PCR must be strictly designed and optimized, and the actual implementation difficulty is high.

Disclosure of Invention

The invention improves the reverse transcription system of RT-qPCR, adopts a brand-new qPCR amplification strategy, can integrate all reactions in one reaction system, and greatly improves the sensitivity when being applied to the detection of mRNA expression level from low-quality and/or a small amount of paraffin tissue sources.

Aiming at the problem of insufficient detection sensitivity of the conventional real-time quantitative PCR in the detection of the mRNA expression level of the paraffin tissue section sample stored for a long time, the invention designs and implements a strategy based on different reverse transcriptions and PCR amplifications, and the reaction steps comprise:

reverse transcription reaction realized by multiple transcription primers with protective neck ring structures;

the external amplification reaction is realized by a 5 'end gene specific primer and a 3' end general external amplification primer; and

nested in-amplification qPCR reactions achieved with 5 'and 3' end-gene specific primers.

Compared with the traditional reverse transcription qPCR, the method not only ensures the specificity of the reverse transcription and qPCR reaction, but also reduces the concentration of the substrate required by the reaction, can improve the detection sensitivity of the qPCR by more than 500 times, and is particularly suitable for the quantitative detection of the mRNA extracted from the paraffin tissue section with low quality and/or low concentration. The method of the invention can greatly improve the sensitivity and reliability of such detection.

Therefore, the present invention provides a novel real-time quantitative PCR method. The method can greatly improve the detection sensitivity of real-time quantitative PCR and the like, and is particularly suitable for extracting RNA from paraffin tissue sections with serious degradation and/or less sample amount.

The real-time quantitative PCR method according to the present invention is a real-time quantitative PCR method particularly suitable for gene expression quantification of FFPE samples, and the reaction steps thereof can be divided into three stages: 1) a reverse transcription stage; 2) a pre-amplification stage; 3) nested in-amplification qPCR stage. In particular, all the reactions may be carried out in one reaction system.

The specific reaction steps are illustrated as follows:

1) reverse transcription reaction: using a fragment of mRNA to be detected (particularly mRNA from an FFPE sample) as a template, synthesizing a first strand cDNA having a stable stem-loop structure at the 5' end by an extension reaction from the 3' end to the 5' end of the template by a reverse transcription primer under the action of a reverse transcriptase, wherein the reverse transcription primer is a single-stranded DNA having a thermostable stem-loop structure consisting of three structural regions: i) a 3' end specific substrate binding region which can match an annealing region bound to the substrate mRNA, preferably consisting of 8 to 15 bases, more preferably consisting of 10 to 12 bases; ii) a stem structural region which is a double-stranded stable structure consisting of a DNA sequence of which the number of bases and the base sequence are perfectly complementary matched, preferably consisting of 6 to 12 bases; and iii) a loop structure region, the sequence of which is located between two DNA sequences forming a stem structure, preferably consisting of 18-25 bases, which does not form any form of stable matched double stranded structure with the i) region and ii) region. Furthermore, when multiple reverse transcription primers are involved for the same mRNA to be detected, these reverse transcription primers have the same sequence of the stem-loop structure region except for the 3' -end specific substrate binding region.

2) External amplification PCR reaction: taking first strand cDNA obtained by reverse transcription reaction as a template, carrying out amplification reaction under the action of DNA polymerase by using a 5' end gene specific primer and a 3' end universal external amplification primer to obtain an external amplification product (the product is double-stranded DNA), wherein the 5' end gene specific primer is matched with the 3' end of the first strand cDNA and is also a 5' end gene specific primer of next nested internal amplification qPCR reaction; the sequence of the 3' end universal external amplification primer is consistent with the loop structure region of the reverse transcription primer in the step 1). Preferably, the 3' universal amplimer primers contain at least one deoxyuridine triphosphate (dUTP). Preferably, the length of the 5 'end gene specific primer and the 3' end universal amplimer is in the range of 10-100 bases, preferably 18-25 bases.

3) Nested in-amplification qPCR reactions: and (3) carrying out internal amplification PCR reaction under the action of DNA polymerase by using a 5 'end gene specific primer and a 3' end gene specific primer as amplification primers and an external amplification product as a template, wherein the 5 'end gene specific primer is the same as the 5' end gene specific primer in the step 2), the 3 'end gene specific primer is positioned at the downstream of the 3' end universal primer of the external amplification PCR, and the gene specificity is matched with a substrate sequence. In some particular embodiments of the invention, 10-20 bases from the 5' end of the 3' end gene-specific primer may coincide with the 3' specific substrate binding region of the reverse transcription primer. In particular, the internal amplification reactions described in the present invention fall within the category of Taqman quantitative PCR.

In step 1) of the method of the present invention, the sequence of the substrate binding region of the reverse transcription primer of the present invention is the 3' -end specific substrate binding region, and preferably, may coincide with the 5' -end sequence of the later-described 3' -end gene-specific primer. The number of reverse transcription primers according to the present invention is generally at least two for one mRNA to be detected, but is not limited to the number. The reverse transcription primers can be designed in a head-to-tail connection mode, namely the 3' end of the second reverse transcription primer is close to the 5' end of the substrate binding region at the 3' end of the first reverse transcription primer; it may also be spaced, i.e.one or more bases apart between the substrate binding regions of the two reverse transcription primers.

In step 1) of the method described herein, the reverse transcription primer is preferably composed of at least two single strands of DNA directed against an mRNA template in a reverse transcription reaction. The reverse transcription primer may in turn anneal to a substrate mRNA strand. The annealing region of the plurality of reverse transcription primers matches 20 to 100 bases, preferably 40 to 50 bases, at a time from the 5 'end to the 3' end of the mRNA.

In step 2) of the method of the present invention, the number of the universal external amplification primers at the 3' end is generally one. But is not limited to a specific number.

In method steps 2) and 3) of the invention, the DNA polymerase is preferably a thermostable DNA polymerase with 5 'to 3' exonuclease activity, such as Taq polymerase (Taq polymerase) or pfu polymerase (pfu polymerase), for use in the exo-amplification PCR reaction and the nested-endo-amplification qPCR reaction.

In step 3) of the method of the invention, preferably, thermostable Uracil glycosidase (Uracil-N-Glycosylase, UNG), such as Afu Uracil-DNA Glycosylase (Afu UNG), is added for specific degradation of the 3' universal external amplification primers.

By thermostable is meant that the enzyme retains at least 5% of its enzymatic activity at 95 ℃ for more than 30 minutes.

In step 3) of the method of the present invention, the number of the 3' end gene-specific primers is generally one. But is not limited to a specific number. In step 2) of the method of the present invention, it is preferable to introduce deoxyuridine dUTP in the 3' universal amplimer instead of dTTP in the original primer. The number of dUTP introductions is one to all dttps.

In step 3) of the method, a taqman molecular indicator probe with a fluorescent label at the 5 'end and a quenching label at the 3' end is used in the nested amplification qPCR reaction, and the taqman molecular indicator probe consists of 18-30 bases. The molecular beacon probe may incorporate one or more modifications at its 5 'end, 3' end or intermediate sequences. Such as common Minor Groove Binder (MGB) modification at the 3' end of the probe for increasing the annealing temperature of the probe.

The method of the present invention can be applied to one or more mRNA sequence fragments to be detected, if no specific reference is made, the number of primers is described as follows: the quantitative terms "at least two", "one", "plurality" and the like are all the number of primers corresponding to one mRNA substrate. If it corresponds to a plurality of mRNA substrates, this is to be understood as the sum of the corresponding amounts.

The scope of the DNA molecules described in the present invention includes base modifications for changing their annealing temperature or increasing the sequence stability, such as Locked Nucleic Acids (LNA), Bridged Nucleic Acids (BNA), Inverted dT, etc. All modifications to the molecule should be considered as extensions of this patent.

Drawings

FIG. 1 schematically shows the structure of a reverse transcription primer used in the method of the present invention.

FIG. 2 schematically shows the reverse transcription, PCR reaction process of the present invention.

FIG. 3 schematically shows the introduction of dUTP and enzymatic cleavage of the outer amplification primer.

FIGS. 4 and 5 show the results of amplification efficiency of nested quantitative PCR according to the example.

Detailed Description

The principles, reaction components and methods related to the present invention, as well as the related terms of art and related patents and literature references related thereto, are explained in detail below with reference to the accompanying drawings.

The illustrations presented herein are to be regarded as increasing the clarity and understandability of the illustrations and are not limiting to the methods of the invention. Where a definition of a term of interest referred to in a patent, reference, etc., is as set forth herein, it is intended that the definition of the term of interest in this document shall control. Some of the enumerated general descriptions herein, such as "a" or "an," are not limited to only one or only one, but should be understood to include the presence of a plurality or multiple of the named species. If the "one primer" is one of many primer choices which can perform the corresponding enzymatic reaction, it is in accordance with the principle of primer design. If said "an enzyme" is a subclass of a certain class of enzymes that meets the defined criteria.

Terms and definitions interpretation:

the "substrate" refers to the DNA or RNA template itself for a nucleic acid molecule-dependent enzymatic reaction such as a polymerase chain reaction or reverse transcription reaction, which may be derived from biological sample extraction, or may be derived from the product of other reactions or artificially synthesized products. Modified bases exogenous to the primer, such as dUTP, etc., may be included. The substrates described herein include mrnas with low integrity and high degradation, and in particular cases include RNA extracted from formalin-fixed, paraffin-embedded tissue sections or sections thereof. Sources of the substrate properties described in this patent include, without limitation, animal/plant tissue, bacteria or viruses, and environmental samples such as water, air, soil samples, and the like. The biological sample extraction source substrate is not limited to any extraction method and extraction platform and related reagents and kits. In some reaction descriptions, such as reverse transcription reactions, the substrate refers to a single strand of mRNA. In other reaction descriptions, the substrate is any one of the DNA double molecular chains, for example. The PCR is a polymerase chain reaction depending on a DNA template, and is characterized in that a substrate is subjected to series-level amplification in a mode of synthesizing a substrate complementary chain by copying a reaction template. The "reverse transcription" is the process of synthesizing its complementary DNA strand from an RNA substrate by reverse transcriptase, the product molecule being RNA-DNA hybrid strand in nature.

The "5 'end" as used herein refers to the end of the DNA or RNA strand where the terminal nucleotide has a free phosphate group, and the "3' end" refers to the end of the DNA or RNA strand where the terminal nucleotide has a free hydroxyl group; the "upstream" refers to a position relatively close to the 5' end; the term "downstream" refers to a location relatively close to the 3' end.

The general PCR "amplification" as used herein refers to exponential PCR amplification, and refers to the amplification of the product obtained by the 5 'end amplification primer as the product of the 3' end primer amplification of the next PCR cycle. The PCR cycle refers to a reaction process of denaturation, annealing and extension. The reaction conditions described, unless otherwise noted herein, should follow reaction conditions that are already widely used and are well known and well known, such as PCR reaction conditions.

The mechanism of the present invention will now be described in detail with reference to the drawings:

the stem-loop structure of the reverse transcription primer of the present invention is shown in FIG. 1, wherein 101 is a 3' terminal specific substrate binding region capable of specifically recognizing and annealing at a specific temperature to bind to a corresponding matching sequence region of a specific gene. The 3 'end gene-specific primer is located downstream of the reverse transcription primer and is generally comprised of 8-15 bases, in the specific case the 3' end specific substrate binding region is comprised of 10 bases. The number of bases of the 3' -end specific substrate binding region referred to herein is understood to be more than 15 bases or less than 8 bases on the basis of the principle of primer design and is not excluded. 102 is the "stem" portion of the stem-loop structure, and the double-stranded stable structure composed of two DNA sequences whose number of bases and base sequence are perfectly complementary matches is generally composed of 6-12 bases, but is not limited to this length range. The "stem" structure has good thermal stability. It should be noted that the base sequence range of the "stem" structural region covers modifications made to improve the thermal stability of the double-stranded structure, such as LNA, BNA, etc. 103 is the "loop" portion of the stem-loop structure, which is a region protected by the "stem" portion, and generally consists of 18-25 bases, but is not limited in scope. For the functional purpose of the invention, the sequence of three functional regions of the reverse transcription primer stem-loop structure is characterized in that bases of any two regions are reduced and complemented to the maximum extent, and a specific stable stem-loop structure is formed. In the actual reaction, the 3' end specific substrate binding region of the reverse transcription primer binds to the complementary matching region of mRNA, and the stem-loop region cannot bind to any region of the substrate depending on its stable structure.

FIG. 2 is a diagram illustrating the reverse transcription reaction, and the process and mechanism of nested PCR reaction, wherein multiple

reverse transcription primers

201 and 204 anneal to the substrate mRNA, and the first strand cDNA is synthesized by extension reaction from the 3 'end to the 5' end of the substrate by reverse transcriptase. Since reverse transcription does not have 3 'to 5' exonuclease activity, the products of the reverse transcription reaction are in fact mixed products of cDNAs of different lengths, with the ratio of the components depending on the binding capacity of the different primers. For mRNA alone, the reverse transcription efficiency is the sum of the reverse transcription efficiencies of different primers, and the reactions of different reverse transcription primers are complementary rather than competitive. While the stable stem-loop structure minimizes the possibility of non-specific transcription of each reverse transcription primer.

The sequence of the substrate binding region of the reverse transcription primer 201 of the present invention may, in some implementations, coincide with the 5 'end sequence of a later-described 3' end gene-specific primer. The reverse transcription primer of the present invention is generally at least two in number, but is not limited to the number. The primer design region can be end-to-end, that is, the 3' end of the second reverse transcription primer is adjacent to the 5' end of the substrate binding region at the 3' end of the first reverse transcription primer; it may also be spaced, i.e.one or more bases apart between the substrate binding regions of the two reverse transcription primers. Fig. 2 shows the first case.

The structure of the cDNA single strand after annealing the reverse transcription product is characterized in that the 5' end has a stable stem-loop structure, and the 3' end has a binding region of the 5' end PCR primer 206. When the 5 'end PCR primer is extended to the 3' end of the cDNA under the conditions of the subsequent PCR reaction, the "stem" structure is eliminated by virtue of the 5 'to 3' exonuclease activity of the DNA polymerase, thereby exposing the loop region sequence 207 and forming the complementary sequence 208. In the next round of PCR, the 3' universal external amplification primer corresponding to the loop region sequence binds to its complementary sequence 209 for extension. The 5 'end gene specific primer and the 3' end universal external amplification primer react to generate external amplification products with different amplification lengths. Wherein each of the outer amplification products comprises a 3' end-gene specific primer matching sequence that serves as a substrate for the 5' end-gene specific primer and the 3' end-gene specific primer 210 in a nested reaction. As the reaction proceeds, the efficiency of the exo-PCR specific amplification decreases at some stage of the PCR and allows the PCR amplification to escape the exponential amplification stage. Resulting in a decrease in the accuracy of qPCR quantification. In order to ensure that the nested qPCR is always in an exponential amplification state before the threshold, the external amplification PCR must be terminated timely. The strategy adopted by the invention is shown in figure 3, namely, deoxyuridine dUTP is introduced into the universal external amplification primer at the 3' end to replace dTTP in the original primer. The number of dUTP introductions is one to all dttps. And adding a thermostable UNG, which is a uracil glycosidase capable of specifically cleaving the phosphate glycosidic bond of deoxyuridine (Sandigursky, et al., J.biol.chem.275(25): 19146-. The thermal stability means that the enzyme activity is still maintained at 5% to 90% after incubation at 95 ℃ for 30 minutes. The optimum working temperature range for the thermostable enzymes should in principle cover 50 ℃ to 75 ℃. During the PCR reaction, dUTP in the outer amplification primers 302/303 and corresponding amplification product 303 will be gradually degraded, so as to finally achieve the purpose of blocking the outer amplification PCR reaction, while the inner amplification primers are not affected by the outer amplification primers, thereby allowing the inner amplification PCR reaction to be performed only. In the whole PCR process, the reverse transcription primer is a stable stem-loop structure and does not participate in amplification reaction, so that the occurrence of non-specific amplification is avoided.

It should be noted that although the principle of the present invention is to integrate the reverse transcription reaction and the PCR nested reaction into one reaction system. In some embodiments, the reaction may be carried out separately in steps. Such as reverse transcription reaction, external amplification PCR reaction, qPCR reaction, which are independently performed in sequence. Then, for example, reverse transcription reaction and external amplification PCR reaction are carried out simultaneously, and then internal amplification qPCR reaction is carried out. And then, independently performing reverse transcription reaction, and simultaneously performing external amplification PCR reaction and internal amplification qPCR reaction. The qPCR reaction described in the invention includes any PCR real-time quantitative technology branch. If a DNA double-strand nonspecific fluorescent dye SYBR Green is adopted, a fluorescent signal is released after the DNA double-strand is combined, and the quantity of the DNA double-strand molecules is determined by detecting the fluorescent signal after the end of each cycle. As another method based on fluorescent probes, a method is adopted in which one end is labeled with a fluorescent group and the other end is labeled with a quencher group (Taqman probe). The molecular probe binds to a specific PCR product single strand by specific base matching, and in the extension step of the PCR reaction, the fluorescent group of the probe is cleaved by the 5 'to 3' exonuclease activity of the DNA polymerase, thereby exposing the fluorescent signal.

Examples

Example one

Nested Taqman qPCR with dUTP primer introduced. The qPCR amplification curves were plotted by artificially synthesized substrates and gradient dilution, showing the amplification efficiency of nested qPCR amplification.

The materials and methods are as follows:

substrates and primers: the substrate is equal concentration mixture of substrate 1 and substrate 2

Substrate 1(SEQ ID NO:1)

GTCTTCCCCTCCATCGTGGGGCGCCCCAGGCACCAGGGCGTGATGGTGGGCATGGGTCAGAAGGATTCCTATGTGGGCGACGAGGCCCAGAGCAAGAGAGGCATCCTCACCCTGAAGTACCCCATCGAGCAAGCTTCGGTTCACGCAATG

Substrate 2(SEQ ID NO:2)

GTCTTCCCCTCCATCGTGGGGCGCCCCAGGCACCAGGGCGTGATGGTGGGCATGGGTCAGAAGGATTCCTATGTGGGCGACGAGGCCCAGAGCAAGAGAGGCATCCTCACCCTGAAGTACAGCTTCGGTTCACGCAATG

5' primer (SEQ ID NO:3) GTCTTCCCCTCCATCGTG

3' external amplification primer (SEQ ID NO:4) CAUUGCGUGACCGAAGCU

3' internal amplification primer (SEQ ID NO:5) GTACTTCAGGGTGAGGATGC

Probe (SEQ ID NO:6) FAM-AATCCTTCTGACCCATGCCCACC-BHQ

qPCR platform: ABI7500 type real-time fluorescence quantitative PCR system

Substrate concentration and amount: substrate concentration was diluted 10-fold in 6 gradients with an initial concentration of 1 ng/. mu.l and a substrate dose of 1. mu.l.

The kit comprises: the reaction system kit is Semiflu Taqman Gene Expression Master Mix.

UNG enzyme: afu UNG was purchased from NEB.

Reaction components

Components	Concentration/amount/volume
		Reaction buffer	1X
Substrate	1ng-10-5ng
		5' primer	0.5μM
3' universal external amplification primer	0.2μM
		3' internal amplification primer	0.5μM
Probe needle	0.25μM
		UNG	0.2U
Reaction volume	20μl

Reaction conditions

As a result, nested quantitative PCR showed very good amplification efficiency as shown in FIGS. 4 and 5.

Example two

Actually, the expression kurtosis of MYB Proto-Oncogene Like 2(MYBL2) gene relative to beta Actin (ACTB) in RNA samples extracted from FFPE samples stored for 8 years is detected. The traditional one-step method qPCR is used as a contrast, and shows that the method is applied to detecting the high sensitivity of the low-quality FFPE sample.

Materials and methods

Substrates and primers

Sample(s)

5 pieces of mammary gland FFPE slices stored for 8 years, Qiagen RNA extraction kit, RNA concentration 25.2 ng/. mu.l, each reaction with RNA 100ng or 10 ng.

Detection of target genes

Substrate 1 beta Actin (ACTB)

Substrate 2 MYB Proto-Oncogene Like 2(MYBL2)

Primers and probes

Reverse transcription primer 1(SEQ ID NO:7)

TCGATAATACGACTCACTATAGGGCATTGCGTGAACCGAAGCCCCTATAGTGAGTCGTATTATCGAGTACTTCAGGGT

Reverse transcription primer 2(SEQ ID NO:8)

TCGATAATACGACTCACTATAGGGCATTGCGTGAACCGAAGCCCCTATAGTGAGTCGTATTATCGAGTGCTCGATGGG

Reverse transcription primer 3(SEQ ID NO:9)

TCGATAATACGACTCACTATAGGGCATTGCGTGAACCGAAGCCCCTATAGTGAGTCGTATTATCGAGGTGACGATGCC

Reverse transcription primer 4(SEQ ID NO:10)

TCGATAATACGACTCACTATAGGGCATTGCGTGAACCGAAGCCCCTATAGTGAGTCGTATTATCGAGTCGTCCCAGTT

Reverse transcription primer 5(SEQ ID NO:11)

TCGATAATACGACTCACTATAGGGCATTGCGTGAACCGAAGCCCCTATAGTGAGTCGTATTATCGACTTTCTCCTGCA

Reverse transcription primer 6(SEQ ID NO:12)

TCGATAATACGACTCACTATAGGGCATTGCGTGAACCGAAGCCCCTATAGTGAGTCGTATTATCGAGGAGCTGCCGGG

Reverse transcription primer 7(SEQ ID NO:13)

TCGATAATACGACTCACTATAGGGCATTGCGTGAACCGAAGCCCCTATAGTGAGTCGTATTATCGATCAGGCGGCCCA

Reverse transcription primer 8(SEQ ID NO:14)

TCGATAATACGACTCACTATAGGGCATTGCGTGAACCGAAGCCCCTATAGTGAGTCGTATTATCGATGTGGCTGGGCT

3' terminal Universal external amplification primer (SEQ ID NO:15) CAUUGCGUGAACCGAAGCU

5' end Gene-specific amplification primer 1(SEQ ID NO:16) GTCTTCCCCTCCATCGTG

5' end Gene-specific enhancer 2(SEQ ID NO:17) GAAGAGATGAATCGGAGCCAG

3' end Gene-specific primer 1(SEQ ID NO:18) GTACTTCAGGGTGAGGATGC

3' end Gene-specific primer 2(SEQ ID NO:19) GCTGGGAGGTAACATGAAGG

Probe 1(SEQ ID NO:20) FAM-AATCCTTCTGACCCATGCCCACC-BHQ

Probe 2(SEQ ID NO:21) VIC-CACCCACAGCAATGAGAAGCACG-TAMRA

The kit comprises: the one-step RT-qPCR Kit is QIAGEN OneStep RT-PCR Kit.

UNG enzyme: afu UNG was purchased from NEB.

Reagents and concentrations are shown in the following table, where reverse transcription primer concentrations refer to reverse transcription mixed primer concentrations, and amplification primer and probe concentrations refer to each primer concentration. Each reaction was set up in 3 replicates.

An experiment platform: ABI7500 type real-time fluorescence quantitative PCR system

1 experimental group

2 control group

Components	Concentration/amount/volume
		QIAGEN one-step RT-PCR buffer solution	1X
dNTP mixture	400μM
		QIAGEN one-step RT-PCR enzyme mixed solution	2μl
Sample RNA	10ng-1ng
		5' gene specific primer	0.2μM
3' gene specific primer	0.2μM
		Probe needle	0.25μM
Reaction volume	20μl

Reaction conditions are as follows:

fluorescence signal channel selection of FAM and VIC

The PCR cycles were set as:

resultant CT value

ACTB

	100ngRNA(±STD)	10ngRNA(±STD)
			Experimental group	19.1±0.4CT	22.4±0.6CT
Control group	25.7±0.5CT	29.2±0.9CT

MYBL2

	100ngRNA(±STD)	10ngRNA(±STD)
			Experimental group	25.6±0.3CT	29.1±0.4CT
Control group	35.2±1.2CT	-

The contrast experiment proves that the method using the RT-qPCR has better reliability and higher sensitivity than the traditional one-step RT-qPCR.

SEQUENCE LISTING

<110> Dalian Tai Biotechnology Ltd

<120> real-time quantitative PCR method suitable for gene expression quantification of FFPE sample

<130> DI17-0733-XC37

<160> 21

<170> PatentIn version 3.3

<210> 1

<211> 150

<212> DNA

<213> Artificial sequence

<220>

<223> substrate 1

<400> 1

gtcttcccct ccatcgtggg gcgccccagg caccagggcg tgatggtggg catgggtcag 60

aaggattcct atgtgggcga cgaggcccag agcaagagag gcatcctcac cctgaagtac 120

cccatcgagc aagcttcggt tcacgcaatg 150

<210> 2

<211> 139

<212> DNA

<213> Artificial sequence

<220>

<223> substrate 2

<400> 2

gtcttcccct ccatcgtggg gcgccccagg caccagggcg tgatggtggg catgggtcag 60

aaggattcct atgtgggcga cgaggcccag agcaagagag gcatcctcac cctgaagtac 120

agcttcggtt cacgcaatg 139

<210> 3

<211> 18

<212> DNA

<213> Artificial sequence

<220>

<223> 5' primer

<400> 3

gtcttcccct ccatcgtg 18

<210> 4

<211> 18

<212> DNA

<213> Artificial sequence

<220>

<223> 3' external amplification primer

<400> 4

cauugcguga ccgaagcu 18

<210> 5

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> 3' internal amplification primer

<400> 5

gtacttcagg gtgaggatgc 20

<210> 6

<211> 23

<212> DNA

<213> Artificial sequence

<220>

<223> Probe

<400> 6

aatccttctg acccatgccc acc 23

<210> 7

<211> 78

<212> DNA

<213> Artificial sequence

<220>

<223> reverse transcription primer 1

<400> 7

tcgataatac gactcactat agggcattgc gtgaaccgaa gcccctatag tgagtcgtat 60

tatcgagtac ttcagggt 78

<210> 8

<211> 78

<212> DNA

<213> Artificial sequence

<220>

<223> reverse transcription primer 2

<400> 8

tcgataatac gactcactat agggcattgc gtgaaccgaa gcccctatag tgagtcgtat 60

tatcgagtgc tcgatggg 78

<210> 9

<211> 78

<212> DNA

<213> Artificial sequence

<220>

<223> reverse transcription primer 3

<400> 9

tcgataatac gactcactat agggcattgc gtgaaccgaa gcccctatag tgagtcgtat 60

tatcgaggtg acgatgcc 78

<210> 10

<211> 78

<212> DNA

<213> Artificial sequence

<220>

<223> reverse transcription primer 4

<400> 10

tcgataatac gactcactat agggcattgc gtgaaccgaa gcccctatag tgagtcgtat 60

tatcgagtcg tcccagtt 78

<210> 11

<211> 78

<212> DNA

<213> Artificial sequence

<220>

<223> reverse transcription primer 5

<400> 11

tcgataatac gactcactat agggcattgc gtgaaccgaa gcccctatag tgagtcgtat 60

tatcgacttt ctcctgca 78

<210> 12

<211> 78

<212> DNA

<213> Artificial sequence

<220>

<223> reverse transcription primer 6

<400> 12

tcgataatac gactcactat agggcattgc gtgaaccgaa gcccctatag tgagtcgtat 60

tatcgaggag ctgccggg 78

<210> 13

<211> 78

<212> DNA

<213> Artificial sequence

<220>

<223> reverse transcription primer 7

<400> 13

tcgataatac gactcactat agggcattgc gtgaaccgaa gcccctatag tgagtcgtat 60

tatcgatcag gcggccca 78

<210> 14

<211> 78

<212> DNA

<213> Artificial sequence

<220>

<223> reverse transcription primer 8

<400> 14

tcgataatac gactcactat agggcattgc gtgaaccgaa gcccctatag tgagtcgtat 60

tatcgatgtg gctgggct 78

<210> 15

<211> 19

<212> DNA

<213> Artificial sequence

<220>

<223> 3' end universal external amplification primer

<400> 15

cauugcguga accgaagcu 19

<210> 16

<211> 18

<212> DNA

<213> Artificial sequence

<220>

<223> 5' end Gene-specific amplification primer 1

<400> 16

gtcttcccct ccatcgtg 18

<210> 17

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> 5' end Gene-specific enhancer 2

<400> 17

gaagagatga atcggagcca g 21

<210> 18

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> 3' end Gene-specific primer 1

<400> 18

gtacttcagg gtgaggatgc 20

<210> 19

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> 3' end Gene-specific primer 2

<400> 19

gctgggaggt aacatgaagg 20

<210> 20

<211> 23

<212> DNA

<213> Artificial sequence

<220>

<223> Probe 1

<400> 20

aatccttctg acccatgccc acc 23

<210> 21

<211> 23

<212> DNA

<213> Artificial sequence

<220>

<223> Probe 2

<400> 21

cacccacagc aatgagaagc acg 23

Claims

1. A real-time quantitative PCR method for determining expression kurtosis of MYBL2 gene relative to ACTB gene of FFPE sample, comprising:

1) reverse transcription reaction: using the ACTB gene as substrate 1 and the mRNA fragment of MYBL2 gene from FFPE sample to be detected as substrate 2 as templates, a first strand cDNA having a stable stem-loop structure at the 5' end was synthesized by an extension reaction from the 3' end to the 5' end of the template by reverse transcriptase using a reverse transcription primer consisting of SEQ ID NO: 7. SEQ ID NO: 8. SEQ ID NO: 9. SEQ ID NO: 10. SEQ ID NO: 11. SEQ ID NO: 12. SEQ ID NO:13 and SEQ ID NO:14, the composition is as follows;

2) external amplification PCR reaction: performing an amplification reaction under the action of DNA polymerase by using a first strand cDNA obtained by a reverse transcription reaction as a template through a 5' end gene specific primer and a 3' end universal external amplification primer to obtain an external amplification product, wherein the 5' end gene specific primer is formed by a primer aiming at a substrate 1 and shown as SEQ ID NO:16 and a 5' end gene specific amplification primer 1 as shown in SEQ ID NO:17, and the 5' end gene specific amplification primer 2; the 3' end universal external amplification primer is shown as SEQ ID NO:15 is shown in the figure;

3) nested in-amplification qPCR reactions: carrying out internal amplification PCR reaction under the action of DNA polymerase by using a 5' end gene specific primer and a 3' end gene specific primer as amplification primers and an external amplification product as a template, wherein the 5' end gene specific primer is formed by a primer sequence which is shown as SEQ ID NO:16 and a 5' end gene specific amplification primer 1 as shown in SEQ ID NO:17, and the 3 'end gene specific primer consists of a primer 2 for amplification specific to a 5' end gene as set forth in SEQ ID NO:18 and a primer 1 specific for the 3' end gene and a primer 2 specific for the substrate as shown in SEQ ID NO:19 and wherein FAM-AATCCTTCTGACCCATGCCCACC-BHQ is applied as probe 1 against substrate 1 and VIC-CACCCACAGCAATGAGAAGCACG-TAMRA is applied as probe 2 against substrate 2 in a nested in-amplification qPCR reaction;

the FFPE sample is a formalin-fixed paraffin-embedded tissue section;

wherein, the expression kurtosis of MYBL2 gene relative to ACTB gene is determined by using QIAGEN OneStep RT-PCR Kit as one-step RT-qPCR Kit and ABI7500 type real-time fluorescent quantitative PCR system as experimental platform, and the reaction system is as follows:

components Concentration/amount/volume QIAGEN OneStep RT-PCR buffer 1X dNTP mixed solution 400μM

Components Concentration/amount/volume QIAGEN OneStep RT-PCR enzyme mixture 2μl Sample RNA 10ng-1ng Reverse transcription primer mixture 0.5μM 3' end universal external amplification primer 0.5μM 5' end gene specific primer 0.2μM 3' end gene specific primer 0.2μM Probe needle 0.25μM Afu UNG from NEB 0.2U Reaction volume 20μl

Wherein the concentration of the reverse transcription primer refers to the concentration of the reverse transcription mixed primer, the concentration of the amplification primer and the probe refers to the concentration of each primer and probe,

the reaction conditions are as follows:

the fluorescent signal channel is selected from FAM and VIC,

the PCR cycles were set as:

48℃ 15min 95℃ 2min 95℃ 15s 45 cycle

48℃ 15min 60℃ 45s

。