CN113337585A

CN113337585A - Small RNA quantitative detection method

Info

Publication number: CN113337585A
Application number: CN202110837219.7A
Authority: CN
Inventors: 刘晓雷; 袁新旺; 徐超; 闫媛媛; 刘冠宏
Original assignee: Shanghai Shengzimao Biotechnology Co ltd; Suzhou Yingze Biomedical Technology Co ltd
Current assignee: Suzhou Yingze Biomedical Technology Co ltd
Priority date: 2021-07-23
Filing date: 2021-07-23
Publication date: 2021-09-03

Abstract

The invention provides a small RNA quantitative detection method for non-disease diagnosis and treatment, which comprises the steps of obtaining cDNA by reverse transcription of poly (A) tailed small RNA, and then carrying out real-time quantitative PCR amplification detection on the cDNA to obtain the expression condition of target small RNA, wherein the sequence composition of a reverse transcription primer for reverse transcription is as follows: 5 ' -tag sequence-Oligo dT-complementary sequence-3 ', wherein the complementary sequence is complementary with the 3 ' end base of the target small RNA, and the tag sequence has no stem-loop structure. The invention can improve the specificity, sensitivity and detection efficiency of the small RNA quantitative detection and can realize the synchronous quantitative detection of various target small RNAs.

Description

Small RNA quantitative detection method

Technical Field

The invention relates to the field of molecular biology, in particular to a small RNA quantitative detection method.

Background

Small RNAs include micro RNAs (miRNAs), short interfering RNAs (siRNAs) and a wide variety of other small non-coding RNAs (ncRNAs), typically less than 30 nucleotides in length. They are present in large amounts in the body and have important physiological effects. For example, miRNA is a small non-coding RNA that regulates gene expression at the transcriptional level, and many studies have shown that miRNA plays an important role in a variety of physiological and pathological processes such as cell proliferation, differentiation, apoptosis, development, tumorigenesis, tumor metastasis, and viral infection. Therefore, the method has important application value in scientific research and clinical diagnosis. Researches show that certain miRNA can act on protooncogenes or cancer suppressor genes through a direct or indirect mode so as to participate in the generation and development of tumors, and the content of the miRNA is related to various cancers. Some miRNA can stably exist in blood, and for tumor patients without typical clinical symptoms, without specificity in examination and difficult in diagnosis, whether the expression level of specific miRNA is in a normal range can be detected through simple blood drawing, so that whether the tumor patients suffer from certain tumors can be screened, and complicated and traumatic biopsy can be avoided.

Researchers have conducted a great deal of research in this area and developed various methods for detecting small RNAs. Among them, the reverse transcription real-time fluorescent quantitative PCR method (RT-qPCR method) has the advantages of totally closed single-tube detection, convenient operation, high sensitivity, good specificity, no need of PCR post-treatment, avoidance of cross contamination, easy automation and the like, and is gradually becoming the main method for small RNA quantitative detection. As with the general mRNA detection, the detection of small RNAs also involves two steps, reverse transcription and PCR. However, since the small RNA is generally less than 30 nucleotides in length, if a reverse transcription primer is designed directly for several base sequences at the 3' end of the small RNA to obtain a cDNA complementary to the small RNA as a template for a PCR step, the amplification efficiency and specificity are poor because the template length is too short when designing a PCR primer. Also, it is not possible to design suitable probes for such short templates. In addition, some family members of different small RNAs have very similar sequences, even only one or two bases, so that the detection of small RNAs has high requirements on specificity.

Currently, there are two major methods for quantitative detection of small RNAs:

the first method is to add a poly (A) sequence to the 3' end of the small RNA by using poly (A) polymerase before reverse transcription (this reaction is called tailing in molecular biology), then use oligo dT as reverse transcription primer to reverse-transcribe to obtain cDNA, and then use the cDNA as template to design primer for real-time quantitative PCR detection. This method has an advantage that cDNA can be obtained more conveniently using oligo dT as a reverse transcription primer without designing a special reverse transcription primer. However, because all RNA added with poly (A) tail is reverse transcribed during reverse transcription, specificity is lacked, the reverse transcription efficiency for target sequences is greatly reduced, and nonspecific reverse transcription products generate serious interference for subsequent PCR detection, the optimization of PCR primers is highly required. Furthermore, because the length of poly (A) tail added after the small RNA is uncertain, the length of PCR product is also uncertain, so that the PCR reaction has no fixed Tm value, and the specificity of the PCR reaction can not be judged by the melting curve. The detection result is influenced by the length of the poly (A) tail added to the 3' end of the small RNA and the low efficiency of reverse transcription reaction, so the sensitivity of the detection method is not ideal sometimes. In addition, the existing tailing reaction and reverse transcription reaction need different reaction systems and need to be carried out step by step, the actual operation is more complicated, and the existing kit can cause the problem of poor specificity if tailing and reverse transcription are carried out simultaneously.

The second method is stem-loop method, which uses a specially designed reverse transcription primer with stem-loop structure, in which several bases at 5 ' end are complementary with a downstream sequence to form a stem-loop structure, and several bases at 3 ' end are complementary with 3 ' end of small RNA. Thus, the reverse transcription product is a long cDNA with a stem-loop structure, and then, a primer and a probe are designed according to the sequence of the cDNA to detect the small RNA through PCR. However, since the small RNAs are all relatively short, the length of the segment of the reverse transcription primer complementary to the small RNA at the 3' end thereof can only be 6 to 8 bases, and it is difficult to actually ensure the specificity of reverse transcription, so that the non-specific binding can only be shielded by the designed stem-loop structure, and the stem-loop structure can also have some adverse effects on PCR, since the reverse primer and probe sequences of PCR are all complementary to the sequence of the stem-loop, but since the stem-loop itself has a reverse complementary sequence, the binding of the primer is hindered during annealing, and the binding of the probe is competitively inhibited, thereby significantly reducing the efficiency of PCR. Therefore, in designing PCR primers, the sequence of the forward primer needs to be carefully optimized to obtain a more desirable result in order to ensure the specificity of amplification. Moreover, the design method of the stem-loop reverse transcription primer is complex, different reverse transcription systems need to be prepared aiming at different target miRNAs in the same sample in order to ensure the specificity of the stem-loop reverse transcription primer, and the synchronous detection of multiple target small RNAs cannot be realized.

Disclosure of Invention

The invention aims to provide a novel method capable of improving specificity, sensitivity and detection efficiency of small RNA quantitative detection.

In order to solve the technical problems, the invention adopts the following technical scheme:

the invention provides a small RNA quantitative detection method for non-disease diagnosis and treatment, which comprises the steps of obtaining cDNA by reverse transcription of poly (A) tailed small RNA, and then carrying out real-time quantitative PCR amplification detection on the cDNA to obtain the expression condition of target small RNA, wherein the sequence composition of a reverse transcription primer for reverse transcription is as follows: 5 ' -tag sequence-Oligo dT-complementary sequence-3 ', wherein the complementary sequence is complementary with the 3 ' end base of the target small RNA, and the tag sequence has no stem-loop structure.

Preferably, the reverse transcription primer and the poly (A) tailed small RNA have 12-21 bases complementary with the template (including Oligo dT segment and complementary sequence segment), and the combination efficiency is greatly improved.

Preferably, the length of the complementary sequence is 4-8 bases.

Further preferably, the complementary sequence is 4 bases or 5 bases or 6 bases in length.

If the complementary sequence is too short (e.g., 2-3 bases), it will not provide the specificity required for reverse transcription; if the complementary sequence is too long, the reverse transcription primer ends will form complementarity with the PCR forward primer ends, resulting in serious false positives.

Preferably, the Oligo dT has a length of 6 to 20 bases.

More preferably, the Oligo dT has a length of 6 bases or more, 16 bases or less, still more preferably 12 bases or less, and yet more preferably 10 bases or less.

More preferably, the Oligo dT is 6 bases, 7 bases, 8 bases, 9 bases, 10 bases, 11 bases, 12 bases or 13 bases in length.

Preferably, the length of the tag sequence is 10-150 bases.

More preferably, the length of the tag sequence is equal to or greater than 15 bases, and still more preferably equal to or greater than 20 bases; more preferably, the tag sequence has a length of 120 bases or less, still more preferably 100 bases or less, yet more preferably 80 bases or less, and still more preferably 60 bases or less.

Preferably, the tag sequence has a length of 20 bases, 21 bases, 22 bases, 23 bases, 24 bases, 25 bases, 26 bases, 27 bases, 28 bases, 29 bases, 30 bases, 31 bases, 32 bases, 33 bases, 34 bases, 35 bases, 36 bases, 37 bases, 38 bases, 39 bases, 40 bases, 41 bases, 42 bases, 43 bases, 44 bases, 45 bases, 46 bases, 47 bases, 48 bases, 49 bases, 50 bases.

The label sequence is a determined sequence generated by a random sequence generation tool.

Preferably, the tag sequence is not complementary to all sequences in the sample to be tested.

Specifically, the tag sequences are aligned using the NCBI blast tool (website address https:// blast. NCBI. nlm. nih. gov/blast. cgi), without returning sequences with a similarity higher than 20%, further preferably without returning sequences with a similarity higher than 10%, and still further preferably without returning sequences with a similarity higher than 5%.

Most preferred tag sequences do not return similar sequences when aligned using the NCBI blast tool.

Preferably, the GC content of the tag sequence is 40-60%, and the distribution is uniform.

More preferably, the GC content of the tag sequence is 42% or more, still more preferably 45% or more, still more preferably 48% or more, and still more preferably 50% or more; more preferably, the GC content of the tag sequence is not more than 58%, still more preferably not more than 55%, and still more preferably not more than 53%.

The tag sequence does not bind to any sequence in a sample to be detected, and the segment serves for subsequent PCR detection and provides a template for PCR primer probe design. And different probes can be designed by adopting different tag sequences at the 5' end, so that simultaneous detection of a plurality of small RNAs can be realized, while the prior art cannot realize simultaneous detection of a plurality of small RNAs.

According to some embodiments, the tag sequence is 15 to 100 bases in length, the Oligo dT is 8 to 15 bases in length, and the complementary sequence is 4 to 6 bases in length.

More preferably, the tag sequence is 20 to 50 bases in length, the Oligo dT is 8 to 15 bases in length, and the complementary sequence is 4 to 6 bases in length.

Preferably, the sequence of the reverse primer for the real-time quantitative PCR amplification detection is designed according to the tag sequence.

Further preferably, the primers of the PCR can be optimized according to the sequence of the small RNA to be detected, so that the reverse primer and the forward primer can be well matched, the Tm values of the two PCR primers are as close as possible, the dimer formation probability is reduced, and the sensitivity and specificity of the PCR detection are greatly improved.

Preferably, the sequence of the reverse primer used for said real-time quantitative PCR amplification detection comprises all or part of said tag sequence.

According to some embodiments, the sequence of the reverse primer used in the real-time quantitative PCR amplification assay is identical to the tag sequence or a segment of the tag sequence.

Preferably, the sample to be detected comprises total RNA, purified small RNA or synthetic small RNA.

Small RNAs detectable by the methods of the present invention include one or more of micro RNAs (miRNAs), short interfering RNAs (siRNAs), and other small non-coding RNAs (ncRNAs).

The methods of the invention can detect small RNAs of less than 30 nucleotides in length.

More preferably, the detection method of the present invention is used for the quantitative detection of miRNA. Specifically, according to some embodiments, the method for quantitatively detecting small RNA specifically comprises:

(1) adding a section of poly (A) sequence to the 3' end of the small RNA in the sample to be detected under the action of poly (A) polymerase to obtain poly (A) tailed small RNA;

(2) reversely transcribing the poly (A) tailed small RNA obtained in the step (1) under the action of the reverse transcription primer to form cDNA;

(3) and (3) carrying out real-time quantitative PCR amplification detection on the cDNA obtained in the step (2) by adopting a dye method or a Taqman probe method to obtain the expression condition of the target small RNA.

More specifically, the reaction system of step (1) is different from the reaction system of step (2).

Preferably, the reaction system of step (1) comprises total RNA, poly (A) polymerase, ATP, poly (A) tailing buffer.

According to one embodiment, the poly (A) tailing buffer packetComprises 250mM NaCl, 50mM Tris-HCl and 10mM MgCl₂The pH was 7.9 at room temperature.

Preferably, the reaction system of step (2) comprises RNA to which a poly (A) tail is added, a reverse transcription primer, a reverse transcriptase, a reverse transcription buffer, dATP, dTTP, dCTP, dGTP.

According to one embodiment, the reverse transcription buffer comprises 50mM Tris-Acetate (pH 8.3), 75mM K Acetate, 3.1mM Mg (OAc)₂2mM DTT, 0.5mM dNTPs each. More specifically, the reaction condition of the step (1) is 35-40 ℃ and 20-40 min;

further preferably, the reaction condition of the step (1) is 36-38 ℃ and 25-35 min;

more specifically, the reaction conditions in the step (2) are 37-52 ℃ and 20-40 min;

further preferably, the reaction condition of the step (2) is 42-51 ℃ and 25-35 min.

More preferably, the reaction condition of the step (2) is 49-51 ℃ and 25-35 min.

Preferably, the sequence of the probe used in the Taqman probe method is designed according to the tag sequence, and if a plurality of target small RNAs are detected simultaneously, the tag sequences at the 5' ends of different reverse transcription primers are different.

More specifically, the probe sequence is identical to a segment of the tag sequence.

At present, in the prior art, tailing and reverse transcription are carried out in different reaction systems step by step, and the tailing and reverse transcription are generally considered to have specificity problems in the same reaction system, because enzymes, substrates, buffers and reaction temperatures adopted by tailing reaction and reverse transcription are different. When tailing and reverse transcription are carried out in the same reaction system after the reverse transcription primer is used, even if the reaction temperature is different, good specificity and reverse transcription efficiency can be guaranteed.

Specifically, according to other embodiments, the poly (a) tailing and the reverse transcription are performed in the same reaction system, and the method for quantitatively detecting the small RNA specifically comprises:

(1) preparing a tailing and reverse transcription reaction system, wherein the tailing and reverse transcription reaction system comprises total RNA, poly (A) polymerase, ATP, reverse transcription primers, reverse transcriptase, reverse transcription buffer, dATP, dTTP, dCTP and dGTP;

(2) the tailing adding and reverse transcription reaction system is reacted for 20-80 min at 35-40 ℃, or is reacted for 20-40 min at 35-40 ℃ and then is reacted for 20-40 min at 40-52 ℃ to obtain cDNA;

Preferably, the tailing and reverse transcription reaction system in the step (2) is reacted for 40-80 min at 37-40 ℃.

Preferably, the tailing and reverse transcription reaction system in the step (2) is reacted for 20-40 min at 35-40 ℃ and then reacted for 20-40 min at 42-51 ℃.

Further preferably, the tailing and reverse transcription reaction system in the step (2) is reacted for 20-40 min at 35-40 ℃ and then for 20-40 min at 49-51 ℃.

More specifically, the reverse transcription buffer used in the same reaction system is mixed with the buffer in step (2) and the total RNA, poly (A) polymerase, ATP, reverse transcription primer, reverse transcriptase are mixed with the reverse transcription buffer at the same time. When the reverse transcription kit is prepared, the concentration of each component can be adjusted according to needs, or other reverse transcription buffer solutions in the existing reverse transcription kit are used.

Specifically, the sequence of the probe used by the Taqman probe method is designed according to the tag sequence.

When only one small RNA is to be quantitatively detected, a dye method or a Taqman probe method can be used.

Preferably, the dye is one or more of SYBR Green, EvaGreen, SYTO-9, SYTO-13, SYTO-16, SYTO-60, SYTO-64, SYTO-82.

Preferably, the Taqman probe sequence is designed according to the tag sequence, and the length of the Taqman probe is preferably 10-20 bases; preferably, the 5 'end of the Taqman probe is labeled by FAM, TAMRA, HEX, VIC, TET, JOE, NED, Cy3, Cy5 and Cy5.5, and the 3' end of the Taqman probe is labeled by MGB, BHQ1, BHQ2, BHQ3 and TAMRA.

When two or more small RNAs need to be detected, different small RNAs are designed into different label sequences, different probe sequences are designed according to the label sequences, and different markers are used to realize simultaneous quantitative detection of multiple small RNAs.

The second aspect of the invention also provides a small RNA quantitative detection kit, which comprises a reverse transcription reaction reagent and a PCR reaction reagent,

the reverse transcription reaction reagent contains a reverse transcription primer, and the sequence composition of the reverse transcription primer is as follows: 5 ' -tag sequence-Oligo dT-complementary sequence-3 ', wherein the complementary sequence is complementary with the base at the 3 ' end of the target small RNA, the tag sequence has no stem-loop structure,

the PCR reaction reagent contains a forward primer and a reverse primer,

the sequence of the forward primer is complementary with the reverse transcribed sequence of the target small RNA,

the sequence of the reverse primer includes all or part of the tag sequence.

Preferably, the small RNA quantitative detection kit further comprises a poly (A) tailing reaction reagent.

Preferably, the small RNA quantitative detection kit further comprises a standard substance of the target small RNA.

Preferably, the small RNA quantitative detection kit further comprises a dye or a probe, and the probe is designed according to the tag sequence.

According to a specific embodiment, the small RNA quantitative detection kit comprises poly (A) polymerase, reverse transcription primer, reverse transcriptase, reverse transcription buffer, ATP, dATP, dTTP, dCTP, dGTP, PCR reaction liquid, forward primer and reverse primer, wherein the PCR reaction liquid contains dye or probe.

In the invention, the Ct value detected by qPCR is used for carrying out relative quantification or absolute quantification on the target small RNA, thereby reflecting the expression condition of the target small RNA.

When the reverse transcription primer is combined with an RNA template, the poly (A) tail and reverse transcription are added simply, the length of a complementary sequence of the stem-loop primer is lengthened, the annealing temperature is increased, and the specificity and the reverse transcription efficiency are effectively improved; the cDNA obtained by the method has a long sequence, the length can reach about 50-150 bases, PCR primers and probes with good specificity can be designed relatively easily, and the PCR detection efficiency and specificity are higher. Furthermore, different probes can be designed by adopting different tag sequences at the 5' end, so that simultaneous detection of a plurality of small RNAs can be realized. In addition, the invention can also ensure good specificity and inversion rate efficiency by carrying out tailing reaction and reverse transcription reaction in the same system.

Compared with the prior art, the invention has the following advantages:

the specificity and sensitivity of the method for detecting the small RNA are obviously superior to those of the existing method, and the accuracy of the method is higher.

The invention further can realize the simultaneous detection of multiple small RNA expression conditions in a sample in one PCR reaction system, thereby greatly reducing the workload of detection personnel and improving the detection efficiency.

The invention can ensure good specificity and inversion rate efficiency even if tailing reaction and reverse transcription reaction are carried out in the same system. The reverse transcription efficiency and the PCR detection efficiency of the invention are higher, so the comprehensive detection efficiency is higher, the detection cost can be obviously reduced, and the invention is worthy of being popularized and utilized in a large range.

Drawings

FIG. 1 is a schematic diagram of the method for quantitatively detecting small RNA according to the present invention, in which the number of A may be changed and the number of T may be changed;

FIG. 2 is an amplification curve detected by the hsa-miR-21-3p dye method in example 1;

FIG. 3 is a melting curve detected by the hsa-miR-21-3p dye method in example 1;

FIG. 4 shows the comparison of the dye method for detecting hsa-miR-21-3p in example 2 and comparative example 1 (amplification curve);

FIG. 5 shows the comparison (melting curve) of the dye method for detecting hsa-miR-21-3p in example 2 and comparative example 1;

FIG. 6 is an amplification curve of comparative example 2;

FIG. 7 is a melting curve of comparative example 2;

FIG. 8 is a standard curve prepared by the method of example 2 in example 3;

FIG. 9 is a calibration curve prepared by the method of comparative example 1 in example 3;

FIG. 10 is an amplification curve detected by the hsa-miR-21-3p probe method in example 4;

FIG. 11 is an amplification curve for simultaneous detection of hsa-miR-21-3p and hsa-miR-375-3p by the probe method in example 5.

Detailed Description

The principle of the invention is shown in figure 1:

first, poly (A) polymerase (which may be from E.coli or other species), ATP, and a sequence of poly (A) are added to a small RNA solution or total RNA containing small RNA, reacted in a suitable buffer, and added to the 3' end of the small RNA.

Then, reverse transcription primers are designed according to the sequence of the small RNA to be detected. The sequence composition of the reverse transcription primer is as follows: 5 '-tag sequence-Oligo dT-complement-3'. Wherein the tag sequence is not combined with the small RNA and has no stem-loop structure; oligo dT is complementary to the tailed poly (A) stretch of small RNA; the complementary sequence is a sequence complementary to the 3' -terminal base of the small RNA to be detected. Thus, the reverse transcription primer can be specifically combined on the target small RNA, Oligo dT is combined with the small RNA, the pairing stability is also increased, and the reverse transcription efficiency is improved.

Then, small RNA added with poly (A) sequence is used as a template, and reverse transcription is carried out by using the reverse transcription primer designed in the previous step to obtain cDNA.

The two reactions of adding the poly (A) tail and reverse transcription may be performed in different reaction systems or in the same reaction system.

And finally, designing primers and probes for PCR reaction according to the cDNA sequence, and carrying out real-time quantitative PCR detection.

In the tailing reaction, the length of poly (A) tail added to the 3' end of the small RNA is uncertain, in the prior art, Oligo dT is used as a reverse transcription primer to obtain various cDNAs (with T of different lengths), the sequence of a PCR product is not single, so that the PCR reaction has no fixed Tm value, and the specificity of the PCR reaction cannot be judged through a melting curve. And all RNAs with poly (A) tails are used as templates for reverse transcription to participate in reverse transcription, so that during reverse transcription, all RNAs such as mRNA, small tailed RNA, rRNA and the like may be subjected to reverse transcription, the reverse transcription efficiency aiming at a target sequence is obviously greatly reduced, and a non-specific reverse transcription product also generates serious interference on subsequent PCR detection, so that the specificity of the prior art is poor.

In the prior art, the reverse transcription 5 ' end used by the stem-loop method is of a stem-loop structure, only 6-8 bases at the 3 ' end are complementary with a template, the binding efficiency is low, and the non-specific binding cannot be obviously reduced, so that the stem-loop method can only shield the non-specific binding through the stem-loop structure at the 5 ' end. However, the stem-loop structure has adverse effect on PCR detection, in the prior art, reverse primer and probe sequences of PCR are complementary with the sequence of the stem-loop, and the stem-loop has a reverse complementary sequence, so that the combination of the primer is blocked during annealing, the combination of the probe is competitively inhibited, and the PCR efficiency is low.

By designing the specific reverse transcription primer, a section of specific sequence is arranged behind the Oligo dT sequence, the number of bases complementary with the template is increased, the binding efficiency is improved, the reverse transcription specificity is greatly improved, the detection result is more accurate, the cDNA generated by reverse transcription almost comes from small RNA to be detected, the amount of the template participating in reverse transcription is greatly reduced, the reverse transcription reaction can be fully carried out, and the efficiency is obviously improved. In addition, as the stem-loop structure influencing PCR reaction is not adopted, the PCR efficiency is obviously higher, and the detection efficiency is higher.

The reverse transcription primer is designed according to a target sequence, is a specific primer, is specifically combined with small RNA added with poly (A) tail, does not need a stem-loop structure, can fully optimize a tag sequence at the 5' end according to needs, enables the reverse primer and the forward primer to achieve good matching, enables the Tm values of the two PCR primers to be as close as possible, reduces the dimer formation probability, and greatly improves the sensitivity and specificity of PCR detection. And the 5' end adopts different label sequences, which can improve the specificity and realize the simultaneous detection of a plurality of small RNAs, which can not be realized by the prior art.

In the prior art, tailing and reverse transcription are carried out in different reaction systems, and the tailing and reverse transcription are generally considered to have specificity problems in the same reaction system, because poly (A) polymerase is used during tailing, ATP and RNA are used as substrates, and the reaction is generally carried out at 37 ℃; reverse transcription is performed by using reverse transcriptase, RNA template, primer and dNTP as substrates, and the reaction is generally performed at 42 ℃, and two reactions with different buffers are required. When tailing and reverse transcription are carried out in the same reaction system after the reverse transcription primer is used, even if the reaction temperature is different, good specificity and reverse transcription efficiency can be guaranteed.

The present invention will be further described with reference to the following examples. However, the present invention is not limited to the following examples. The implementation conditions adopted in the embodiments can be further adjusted according to different requirements of specific use, and the implementation conditions not mentioned are conventional conditions in the industry. The technical features of the embodiments of the present invention may be combined with each other as long as they do not conflict with each other. The raw materials, reagents and the like used in the examples of the present invention are commercially available.

The following embodiments are provided to illustrate specific embodiments of the present invention:

example 1

Detection of hsa-miR-21-3p (tailing and reverse transcription are carried out in different reaction systems, dye method)

Sample preparation: total RNA was extracted from 100 million 293T cells using TRIzol reagent, dissolved in 100. mu.l of water, and the concentration was measured for use. The total RNA sample contains small RNA, and is used as a sample to be detected in the embodiment of the present invention. RNA sequence and primer design:

the sequence of hsa-miR-21-3p is 5'-caacaccagucgaugggcugu-3' (SEQ ID No. 1).

The reverse transcription primer hsa-miR-21-RT sequence designed according to the last base sequences is as follows:

5’-gttactctgtagcacggatctccttttttttacagc-3’(SEQ ID No.2)。

the 23 bases at the 5' end of the sequence are a label sequence generated by using a random sequence generation tool, the GC content is 52.2 percent, the distribution is uniform, and no similar sequence is found by using NCBI blast tool (the website is https:// blast. The middle is 8T, and the last 5 bases are reversely complementary with the 5 bases at the 3' end of the small RNA to be detected. Designing PCR primers according to the sequence of the reverse transcription product,

the sequence of the forward primer hsa-miR-21-3p-F is 5'-gggcaacaccagtcgatg-3' (SEQ ID No. 3).

The sequence of the reverse primer hsa-miR-21-3p-R is 5'-gttactctgtagcacggatctcc-3' (SEQ ID No. 4).

RNA plus poly (a) tail:

1 microgram of total RNA was taken,

adding 0.5U of poly (A) polymerase (from Escherichia coli, NEB cat # M0276S),

1 microliter of 10mM ATP, in the presence of water,

1 microliter of 10 Xbuffer (as supplied by M0276S),

adding water to 10 microliter, mixing, cooling at 37 deg.C for 30 min, and freezing.

Reverse transcription:

taking 5 microliter of RNA added with poly (A) tail (namely the reaction product of the previous step),

adding 1 microliter hsa-miR-21-RT primer (the concentration is 4 MuM),

1 microliter of reverse transcriptase (Invitrogen cat # 18080-093),

4 microliters of 5 XTRT Buffer (5 XTIRE-Strand Buffer in Invitrogen kit),

1 microliter of 10mM dNTP mixture,

adding water to 20 microliter, mixing evenly,

the cDNA obtained by reverse transcription was placed on ice at 50 ℃ for 30 minutes for future use.

Real-time quantitative PCR:

20 microliters of cDNA was diluted with water to 100 microliters and used as a template for the PCR reaction. A10 microliter PCR system was prepared according to the instructions of the Applied Biosystems Power Up SYBR Green Master Mix (cat # A25741), with 2 microliter template, and 2 microliter primers using hsa-miR-21-3p-F, hsa-miR-21-3p-R, and expression of hsa-miR-21-3p was detected using the Applied Biosystems ViiA7 qPCR instrument. As a result of the test, the amplification curve is shown in FIG. 2, and it can be seen from FIG. 2 that the Ct value is 28.2, the melting curve is shown in FIG. 3, and FIG. 3 shows that the melting curve is a single peak, indicating that the detection method of this example has good specificity.

Example 2

Detection of hsa-miR-21-3p (tailing reaction and reverse transcription are carried out in the same reaction system, dye method)

The hsa-miR-21-RT primers and the PCR primer hsa-miR-21-3p-F, PCR primer hsa-miR-21-3p-R are the same as in example 1.

Tailing reverse transcription reaction:

1 microgram of total RNA was taken,

adding 0.5U of poly (A) polymerase (from Escherichia coli, NEB cat # M0276S),

2 microliters of 10mM ATP was added to the sample,

1 microliter hsa-miR-21-RT primer (the concentration is 4 MuM),

1 microliter of reverse transcriptase (Invitrogen cat # 18080-093),

4 microliters of 5 XTRT Buffer (5 XTIRE-Strand Buffer in Invitrogen kit),

1 microliter of 10mM dNTP mixture,

adding water to 20 microliter, mixing, carrying out tailing reaction at 37 deg.C for 30 min,

then, reverse transcription was performed at 50 ℃ for 30 minutes.

The resulting cDNA was then placed on ice for use.

Real-time quantitative PCR:

20 microliters of cDNA was diluted with water to 100 microliters and used as a template for the PCR reaction. A10 microliter PCR system was prepared according to the instructions of the Applied Biosystems Power Up SYBR Green Master Mix (cat # A25741), with 2 microliter template, and 2 microliter primers using hsa-miR-21-3p-F, hsa-miR-21-3p-R, and expression of hsa-miR-21-3p was detected using the Applied Biosystems ViiA7 qPCR instrument. As a result of the test, the amplification curve was shown in FIG. 4, in which the Ct value was 27.056, and the melting curve was shown in FIG. 5, in which the melting curve was a single peak, indicating that the detection method of this example had excellent specificity.

Comparative example 1

Detecting the expression of hsa-miR-21-3p by adopting a stem-loop method:

in the comparative example, the miRNA 1st Strand cDNA Synthesis Kit (by stem-loop) Kit (cat # MR101-01) of Nanjing Novozam is selected to detect the expression of hsa-miR-21-3 p.

Firstly, a reverse transcription primer hsa-miR-21-RT2V with a sequence of 5'-gtcgtatccagtgcagggtccgaggtattcgcactggatacgacagagcc-3' (SEQ ID No.5) is designed according to the instruction of the kit. The last 6 basic groups of the primer are reversely complemented with the last 6 basic groups of hsa-miR-21-3 p; the preceding sequence is the stem-loop sequence recommended in the kit instructions. And designing PCR primers according to the sequence of the reverse transcription product, wherein the sequence of the forward primer hsa-miR-21-3p-F is 5'-GGGCAACACCAGTCGATG-3' (SEQ ID No.3), and the sequence of the reverse primer hsa-miR-21-3p-R2V is 5'-ccagtgcagggtccgaggta-3' (SEQ ID No. 6).

Taking 1 microgram of total RNA, adding a reverse transcription primer, reverse transcriptase, buffer and the like according to the operation of a kit instruction, and carrying out reverse transcription reaction to obtain cDNA. Taking cDNA to do real-time quantitative PCR detection. The PCR primers used hsa-miR-21-3p-F (SEQ ID No.3) and hsa-miR-21-3p-R2V (SEQ ID No.6), and the preparation of the PCR reaction system was the same as the real-time quantitative PCR step of example 1.

The amplification results obtained by the test are shown in FIG. 4, and the Ct value of comparative example 1 is 27.58; compared with example 2, the Ct value of comparative example 1 is larger than that of example 2, which shows that the detection sensitivity of example 2 is higher than that of comparative example 1. The melting curve is shown in FIG. 5, and the melting curve of comparative example 1 is bimodal, indicating that the specificity is not good.

Comparative example 2

Detection of hsa-miR-21-3p (SEQ ID No.1) (poor quality of tag sequence in reverse transcription primer)

A tag sequence 5'-CGGAGGCCCGGAGCGGGGA-3' of 19 nucleotides in length was generated using a random sequence generation tool, the sequence having a GC content of 84.2% and multiple consecutive C or G's therein. This sequence was added with 8T and the last 6 nucleotides complementary to the end of hsa-miR-21-3p to give the reverse transcription primer hsa-miR-21-RT 1C: 5'-cggaggcccggagcggggatttttttttacagc-3' (SEQ ID No. 7). The sequence of the corresponding PCR forward primer hsa-miR-21-3p-F is 5'-GGGCAACACCAGTCGATG-3' (SEQ ID No.3), and the sequence of the reverse primer hsa-miR-21-3p-RC is 5'-cggaggcccggagcgggga-3' (SEQ ID No. 8). The steps of example 2 are followed to carry out tailing reverse transcription and real-time quantitative PCR detection on the expression of hsa-miR-21-3p (SEQ ID No.1), an amplification curve is shown in figure 6, a Ct value is 25.5, a melting curve is shown in figure 7, and obvious double peaks appear in figure 7, so that the specificity is poor, and therefore, the optimization of a tag sequence in a reverse transcription primer is necessary to ensure the specificity of detection.

Example 3

The detection methods of example 2 and comparative example 1 are respectively adopted to detect the hsa-miR-21-3p standard sample:

dissolving the synthesized hsa-miR-21-3p small RNA dry powder with DEPC water, performing gradient dilution to corresponding concentration according to the table 1, taking 4 microliters of each sample, performing tailing reverse transcription according to the method of the embodiment 2 and the method of the comparative example 1 respectively, and performing real-time quantitative PCR detection, wherein the Ct value is shown in the table 2.

A standard curve is plotted with the Ct value in Table 2 as the ordinate and the log value of the concentration of the standard sample as the abscissa, and the standard curve prepared by the quantitative method of example 2 is shown in FIG. 8, and the standard curve prepared by the quantitative method of comparative example 1 is shown in FIG. 9.

TABLE 1 dilution of RNA standards

Rod numbering	Concentration (mol/L)
		1	1×10^-10
2	2.5×10^-11
		3	6.25×10^-12
4	1.56×10^-12
		5	3.91×10^-13
6	9.77×10^-14
		7	2.44×10^-14
8	6.1×10^-15

TABLE 2 real-time quantitative PCR results

As can be seen from Table 2 and FIG. 8, the Ct values detected by the method provided in example 2 increased with decreasing sample concentration, and they maintained a good logarithmic relationship (see FIG. 8 for the fitting results, R)²0.9989), Ct value was 27.056 in real-time quantitative PCR assay in example 2, and hsa-miR-21-3 was calculated from total RNA detected by the method of example 2The concentration of p is 1.6X 10^-13mol/L。

As is clear from Table 2 and FIG. 9, the Ct value detected by the method of comparative example 1 also increased with decreasing sample concentration, but the correlation was significantly lower than that of example 2 (see FIG. 9 for the fitting results, R)²＝0.9891)。

According to Table 2, the Ct value measured using the method of comparative example 1 was larger than that measured using the method of example 2 at the same concentration when the concentration of the small RNA sample was high, but when the concentration of the small RNA sample was low (2.44X 10)^- ¹⁴mol/L、6.1×10^-15mol/L), the Ct value detected by the comparative example 1 is not much different from the Ct value detected by the corresponding concentration of the example 2, theoretically, the Ct value detected by using the method of the comparative example 1 should be higher than the Ct value detected by using the method of the example 2 at low concentration, so that the detection technology using the comparative example 1 is probably poor in specificity when the concentration of the small RNA sample is low, non-specific amplification occurs, and the Ct value is smaller.

Example 4

Detection of hsa-miR-21-3p (tailing reaction and reverse transcription reaction are carried out in the same reaction system, probe method)

Primer and probe design:

the reverse transcription primer hsa-miR-21-RT3P sequence used for detecting by the probe method is as follows: 5'-gttactctgtagcacggatctcctctgagctgagtagcagttttttttacagc-3' (SEQ ID No. 9).

The forward and reverse primer sequences for PCR are shown in example 1, hsa-miR-21-3p-F (SEQ ID No.3), hsa-miR-21-3p-R (SEQ ID No. 4);

the sequence of the probe hsa-miR-21-3p-PB is FAM-ctgagctgagtagcag-MGB (SEQ ID No. 10).

Tailing reverse transcription reaction:

1 microgram of total RNA was taken,

adding 0.5U of poly (A) polymerase (from Escherichia coli, NEB cat # M0276S),

2 microliters of 10mM ATP was added to the sample,

1 microliter hsa-miR-21-RT3P primer (the concentration is 4 MuM),

1 microliter of reverse transcriptase (Invitrogen cat # 18080-093),

4 microliters of 5 XTRT Buffer (5 XTIRE-Strand Buffer in Invitrogen kit),

1 microliter of 10mM dNTP mixture,

then, reverse transcription was performed at 50 ℃ for 30 minutes. The resulting cDNA was then placed on ice for use.

Real-time quantitative PCR:

20 microliters of cDNA was diluted with water to 100 microliters and used as a template for the PCR reaction. A10 microliter PCR system was prepared according to the instructions of the Applied Biosystems TaqMan Fast Advanced Master Mix (cat # 4444556) and expression of hsa-miR-21-3p (SEQ ID No.1) was detected using an Applied Biosystems ViiA7 qPCR instrument. The detection results are shown in fig. 10.

Example 5

Simultaneously detecting expression of hsa-miR-21-3p and hsa-miR-375-3p (probe method)

RNA sequence and primer, probe design:

the RNA sequence (SEQ ID No.1) of hsa-miR-21-3p, the reverse transcription primer (SEQ ID No.9), the PCR primer (SEQ ID Nos. 3-4) and the probe hsa-miR-21-3p-PB sequence (SEQ ID No.10) are shown in example 1 and example 4;

the sequence of hsa-miR-375-3p is 5'-uuuguucguucggcucgcguga-3' (SEQ ID No. 11).

The reverse transcription primer hsa-miR-375-RT1P has the sequence as follows:

5’-cgcagatggtgtgtcccgacgttgtgatactctcgctgtgttttttttcacgc-3’(SEQ ID No.12)。

designing PCR primers according to the sequence of the reverse transcription product,

the sequence of the forward primer hsa-miR-375-3p-F is 5'-ggtttgttcgttcggctc-3' (SEQ ID No.13),

the sequence of the reverse primer hsa-miR-375-3p-R is 5'-gcagatggtgtgtcccga-3' (SEQ ID No. 14).

The sequence of the probe hsa-miR-375-3p-PB is TAMRA-ttgtgatactctcgct-MGB (SEQ ID No. 15).

Tailing reverse transcription reaction:

1 microgram of total RNA was taken,

adding 0.5U of poly (A) polymerase (from Escherichia coli, NEB cat # M0276S),

2 microliters of 10mM ATP was added to the sample,

1 microliter each of hsa-miR-21-RT3P (SEQ ID No.9) and hsa-miR-375-RT1P (SEQ ID No.12) (concentration is 4. mu.M),

1 microliter of reverse transcriptase (Invitrogen cat # 18080-093),

4 microliters of 5 XTRT Buffer (5 XTIRE-Strand Buffer in Invitrogen kit),

1 microliter of 10mM dNTP mixture,

then, reverse transcription was performed at 50 ℃ for 30 minutes.

The resulting cDNA was then placed on ice for use.

Real-time quantitative PCR:

20 microliters of cDNA was diluted with water to 100 microliters and used as a template for the PCR reaction. A10 microliter PCR system was prepared according to the instructions of the Applied Biosystems TaqMan Fast Advanced Master Mix (cat # 4444556), and the expression of hsa-miR-21-3p (SEQ ID No.1) and hsa-miR-375-3p (SEQ ID No.11) was simultaneously detected in the same reaction system using an Applied Biosystems ViiA7 qPCR instrument. The detection results are shown in fig. 11. FIG. 11 shows that the Ct values of hsa-miR-21-3p and hsa-miR-375-3p are different, the expression of the two is different, and the Ct value of hsa-miR-375-3p is smaller than that of hsa-miR-21-3p, which indicates that the expression amount of hsa-miR-375-3p is higher relative to hsa-miR-21-3 p. The results show that the detection method of the invention can be used for simultaneously detecting the expression of a plurality of small RNAs in a sample in one PCR reaction system.

The present invention has been described in detail in order to enable those skilled in the art to understand the invention and to practice it, and it is not intended to limit the scope of the invention, and all equivalent changes and modifications made according to the spirit of the present invention should be covered by the present invention.

Sequence listing

<110> Onzhize, Suzhou, Bio-medicine technology, Inc

Shanghai Shengcatalpen Biotechnology Co., Ltd

<120> small RNA quantitative detection method

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<170> SIPOSequenceListing 1.0

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<213> Artificial sequence (rengongxulie)

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gggcaacacc agtcgatg 18

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<212> DNA

<213> Artificial sequence (rengongxulie)

<400> 4

gttactctgt agcacggatc tcc 23

<210> 5

<211> 50

<212> DNA

<213> Artificial sequence (rengongxulie)

<400> 5

gtcgtatcca gtgcagggtc cgaggtattc gcactggata cgacagagcc 50

<210> 6

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<213> Artificial sequence (rengongxulie)

<400> 6

ccagtgcagg gtccgaggta 20

<210> 7

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<213> Artificial sequence (rengongxulie)

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cggaggcccg gagcggggat ttttttttac agc 33

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ctgagctgag tagcag 16

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ggtttgttcg ttcggctc 18

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gcagatggtg tgtcccga 18

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Claims

1. A small RNA quantitative detection method for non-disease diagnosis and treatment purposes, which obtains cDNA through reverse transcription of poly (A) tailed small RNA, and then carries out real-time quantitative PCR amplification detection on the cDNA by adopting a Taqman probe method to obtain the expression condition of target small RNA, and is characterized in that the sequence composition of a reverse transcription primer for reverse transcription is as follows: 5 ' -tag sequence-Oligo dT-complementary sequence-3 ', wherein the complementary sequence is complementary with the base at the 3 ' end of the target small RNA, the tag sequence has no stem-loop structure, the tag sequence is not complementary with all sequences in a sample to be detected, the GC content of the tag sequence is 40% -60%, the sequence of the probe is the same as one section of the tag sequence,

the method for quantitatively detecting the small RNA is used for simultaneously detecting the expression conditions of a plurality of target small RNAs, the tag sequences at the 5' ends of different reverse transcription primers are different, and different probes use different markers.

2. The method for quantitatively detecting small RNA according to claim 1, wherein the length of the complementary sequence is 4 to 8 bases,

and/or the Oligo dT has the length of 6-20 bases,

and/or the length of the tag sequence is 10-150 bases.

3. The method for quantitatively detecting small RNA according to claim 1, wherein the tag sequence is 15 to 100 bases long, the Oligo dT is 8 to 15 bases long, and the complementary sequence is 4 to 6 bases long.

4. The method for the quantitative detection of small RNA according to claim 1, wherein the sequence of the reverse primer used for the real-time quantitative PCR amplification detection comprises all or part of the tag sequence.

5. The method for quantitative determination of small RNA according to claim 1, wherein the sample to be detected suitable for the method for quantitative determination of small RNA comprises total RNA, purified small RNA or synthetic small RNA.

6. The method for the quantitative detection of small RNA according to claim 1, wherein the small RNA comprises one or more of miRNA, siRNA or ncRNA.

7. The method for the quantitative detection of small RNA according to any one of claims 1 to 6, wherein the method for the quantitative detection of small RNA specifically comprises:

(3) and (3) carrying out real-time quantitative PCR amplification detection on the cDNA obtained in the step (2) by adopting a Taqman probe method to obtain the expression condition of the target small RNA.

8. The method for quantitatively detecting a small RNA according to claim 7,

the reaction condition of the step (1) is 35-40 ℃ and 20-40 min;

the reaction condition of the step (2) is 37-52 ℃ and 20-40 min.

9. The method for quantitatively detecting a small RNA according to claim 7,

the reaction system in the step (1) comprises a sample to be detected, poly (A) polymerase, ATP and poly (A) tailing buffer;

the reaction system of the step (2) comprises the sample to be detected after the reaction of the step (1), the reverse transcription primer, reverse transcriptase, a reverse transcription buffer solution, dATP, dTTP, dCTP and dGTP.

10. The method for quantitative detection of small RNA according to any one of claims 1 to 6, wherein poly (A) tailing and reverse transcription are performed in the same reaction system, and the method for quantitative detection of small RNA specifically comprises:

(1) preparing a poly (A) tailing and reverse transcription reaction system, wherein the poly (A) tailing and reverse transcription reaction system comprises a sample to be detected, poly (A) polymerase, a reverse transcription primer, reverse transcriptase, a reverse transcription buffer solution, ATP, dTTP, dCTP, dGTP and dATP;

(2) the tailing and reverse transcription reaction system is reacted for 20-80 min at 35-40 ℃, or is reacted for 20-40 min at 35-40 ℃ and then is reacted for 20-40 min at 40-52 ℃ to obtain cDNA;

11. A small RNA quantitative detection kit is characterized in that the small RNA quantitative detection kit comprises a reverse transcription reaction reagent and a PCR reaction reagent,

the reverse transcription reaction reagent contains a reverse transcription primer, and the sequence composition of the reverse transcription primer is as follows: 5 ' -tag sequence-Oligo dT-complementary sequence-3 ', wherein the complementary sequence is complementary with the base at the 3 ' end of the target small RNA, the tag sequence has no stem-loop structure, and the tag sequence is not complementary with all sequences in a sample to be detected; the GC content of the tag sequence is 40-60%, the PCR reaction reagent contains a forward primer, a reverse primer and a probe,

the sequence of the reverse primer comprises all or part of the sequence of the tag,

the sequence of the probe is identical to a segment of the tag sequence,