CN105039564A - miRNA (microribonucleic acid) detection chip, and manufacturing method and application thereof - Google Patents
miRNA (microribonucleic acid) detection chip, and manufacturing method and application thereof Download PDFInfo
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Abstract
The invention relates to an miRNA (microribonucleic acid) detection chip which comprises a substrate provided with a gold film, a specific RNA (ribonucleic acid) probe and a single-chain DNA (deoxyribonucleic acid) probe, wherein the specific RNA probe contains a specific sequence for detecting the target miRNA, and can be hybridized with cDNA (complementary deoxyribonucleic acid) formed by target miRNA reverse transcription; and the single-chain DNA probe is used as a detection control. The specific RNA probe and single-chain DNA probe are respectively provided with fluorescence markers, the cDNA formed by reverse transcription of the miRNA to be detected and the RNA probe form an miRNA/cDNA hybrid, and ribonuclease is utilized to digest the miRNA in the miRNA/cDNA hybrid, so that the fluorescence markers combined on the specific RNA probe depart; and the initial fluorescence intensity of the chip is compared with the fluorescence intensity after enzyme digestion to obtain the degradation quantity of the specific RNA probe on the miRNA detection chip surface, thereby obtaining the content of the miRNA to be detected. Besides, the invention also relates to a manufacturing method and application of the miRNA detection chip.
Description
Technical field
The present invention relates to biochemistry detection field, especially relate to a kind of miRNA detection chip and preparation method thereof and application.
Background technology
The endogenous non-coding small RNA molecular of microRNA (Microrna, also known as miRNA) to be a kind of length be 18 ~ 22nt, is prevalent in animal and plant body.MicroRNA is regulators of gene expression important in organism, participates in regulating each processes such as early embryo development, cell proliferation and differentiation apoptosis and metabolism, and the generation of its differential expression and multiple human diseases develops closely related.The different steps of various disease has its corresponding miRNA express spectra, and serum/plasma endogenous miRNA is relatively stable.In recent years, miRNA is considered novel biomarker clinically, can provide brand-new important evidence and Research Thinking for the diagnosis of disease, treatment.The main detection method of current miRNA has NorthernBlotting, microarray technology and RT-PCR etc., and these technology exist certain detection defect all separately, as complicated, consuming time in NorthernBlotting method, cost is high and sensitivity is low; RT-PCR has good accuracy, higher sensitivity, can detect low abundance miRNA, but causes amplification technique difficulty because the length of miRNA is shorter than primer and can not high throughput testing; Although microarray technology can realize high throughput testing miRNA, its experiment reagent, plant and instrument are expensive, and cost is high.
Biochip technology disposablely can carry out determination and analysis to sequence a large amount of in sample, is widely used in the field such as qualification, medical diagnosis of gene expression analysis, virus and bacterium.But traditional biochip technology often needs PCR to realize the amplification of signal, operate too loaded down with trivial details, sensitivity is not high, is difficult to the analysis adapting to high-throughout gene.
Summary of the invention
Based on this, be necessary to provide a kind of easy and simple to handle and miRNA detection chip that can realize high throughput testing and preparation method thereof and application.
A kind of miRNA detection chip, comprise be provided with golden film substrate, specific RNA probe and ssDNA probe;
Described specific RNA probe contains the specific sequence for detecting target miRNA, and described specific sequence can be hybridized with the cDNA formed by described target miRNA reverse transcription;
One end modified biological element of described specific RNA probe, described vitamin H is combined with fluorescent marker, the other end of described specific RNA probe modifies sulfydryl, described specific RNA probe modification have one end of sulfydryl be fixed on by Au-S key described in be provided with in the substrate of golden film;
Described ssDNA probe is for detecting contrast, one end modified biological element of described ssDNA probe, described vitamin H is combined with fluorescent marker, the other end of described ssDNA probe modifies sulfydryl, is provided with in the substrate of golden film described in one end that described ssDNA probe is modified with sulfydryl is fixed on by Au-S key.
In one embodiment, described specific RNA probe is a kind of, and the sequence of described specific RNA probe is SEQIDNo.1 or SEQIDNo.2.
In one embodiment, described specific RNA probe is two kinds, and the sequence of described specific RNA probe is respectively SEQIDNo.1 and SEQIDNo.2.
In one embodiment, the sequence of described ssDNA probe is as shown in SEQIDNo.3.
In one embodiment, described fluorescent marker is the streptavidin of marked by fluorescein isothiocyanate.
The making method of above-mentioned miRNA detection chip, comprises the steps:
Build the specific sequence for detecting target miRNA, described specific sequence can be hybridized with the cDNA formed by target miRNA reverse transcription, described specific sequence one end modified biological element and fluorescent marker is combined on described vitamin H, then modify sulfydryl at the other end of described specific sequence, obtain specific RNA probe;
Build single-stranded DNA sequence for detecting contrast, described single-stranded DNA sequence one end modified biological element and fluorescent marker is combined on described vitamin H, then modify sulfydryl at the other end of described single-stranded DNA sequence, obtain ssDNA probe;
Form one deck gold film at substrate surface, obtain the substrate being provided with golden film;
By described specific RNA probe and described ssDNA probe respectively point sample to described in be provided with in the substrate of golden film, 4 DEG C of sealings are preserved, and make described specific RNA probe and between described ssDNA probe and the described substrate being provided with golden film, the self-assembly of Au-S key occur and be fixed probe; And
Adopt ethanol solution to close white space, obtain described miRNA detection chip.
A detection method of miRNA, comprises the steps:
Extract miRNA to be measured, and with the miRNA described to be measured extracted for template, under the effect of reversed transcriptive enzyme, reverse transcription forms the cDNA with described miRNA complementation to be measured;
Carry out fluoroscopic examination under miRNA detection chip described in above-mentioned any embodiment is placed in fluorescent microscope, obtain initial fluorescent intensity;
In described miRNA detection chip, add the cDNA with described miRNA complementation to be measured, obtain miRNA/cDNA crossbred;
Add the miRNA in miRNA/cDNA crossbred described in Yeast Nucleic Acid enzymic hydrolysis, discharge the cDNA in crossbred;
CDNA, rnase and fluorescent marker that washing removing is free, carry out fluoroscopic examination under described miRNA detection chip is placed in fluorescent microscope, obtain enzyme and cut rear fluorescence intensity; And
Cut rear fluorescence intensity by more described initial fluorescent intensity and described enzyme, obtain the degradation amount of the specific RNA probe on miRNA detection chip surface, thus obtain the content of miRNA to be measured.
In one embodiment, described in obtain after enzyme cuts rear fluorescence intensity, also comprise chip regeneration step, described chip regeneration step is the NaOH solution regeneration chip surface adopting 10mmol/L, then rinses chip surface with the aqueous solution of not qiagen rnase enzyme.
In one embodiment, in the operation of the described miRNA added in miRNA/cDNA crossbred described in Yeast Nucleic Acid enzymic hydrolysis, described hydrolysis operation is dripped by rnase on described miRNA detection chip surface, be paved with probe area, leave standstill 10 minutes, make rnase cut miRNA in described miRNA/cDNA crossbred, discharge the cDNA in crossbred, the cDNA discharged continues again the remaining complete miRNA probe chain with chip surface and is hybridized, is hydrolyzed.
In one embodiment, described rnase is RNaseH, and the concentration of described RNaseH is 60U/mL.
Specific RNA probe in above-mentioned miRNA detection chip and ssDNA probe are all combined with fluorescent marker, and specific RNA probe contains the specific sequence for detecting target miRNA.When the cDNA that miRNA reverse transcription to be measured is formed can be hybridized with this specific sequence, form miRNA/cDNA crossbred.The miRNA in miRNA/cDNA crossbred is cut again by use rnase enzyme, the fluorescent marker be combined on specific RNA probe is departed from, rear fluorescence intensity is cut by comparable chip initial fluorescent intensity and enzyme, obtain the degradation amount of the specific RNA probe on miRNA detection chip surface, thus obtain the content of miRNA to be measured.Simultaneously, the cDNA of release can be combined with the specific RNA sequence of chip surface repeatedly, then is hydrolyzed, until the RAN probe of chip surface is hydrolyzed completely, thus define a kind of amplification detection effect based on non-PCR, the high throughput testing that can realize miRNA easy and simple to handle.
Accompanying drawing explanation
Fig. 1 is the structural representation of the miRNA detection chip of an embodiment;
Fig. 2 is the schema of the making method of the miRNA detection chip of an embodiment;
Fig. 3 is the schema of the detection method of the miRNA of an embodiment;
Fig. 4 is the canonical plotting that cDNA concentration and initial fluorescent intensity and enzyme cut rear fluorescence intensity difference;
Fig. 5 a is the initial fluorescent intensity photo detecting ssDNA probe region in miRNA detection chip under the electrically and biologically fluorescent microscope of an embodiment;
Fig. 5 b is that the enzyme detecting ssDNA probe region in miRNA detection chip under the electrically and biologically fluorescent microscope of an embodiment cuts rear fluorescence intensity photo;
Fig. 6 a is the initial fluorescent intensity photo detecting specificity mir-29a-3pRNA probe area in miRNA detection chip under the electrically and biologically fluorescent microscope of an embodiment;
Fig. 6 b is that the enzyme detecting specificity mir-29a-3pRNA probe area in miRNA detection chip under the electrically and biologically fluorescent microscope of an embodiment cuts rear fluorescence intensity photo;
Fig. 7 a is the initial fluorescent intensity photo detecting specificity mir-181a-5pRNA probe area in miRNA detection chip under the electrically and biologically fluorescent microscope of an embodiment;
Fig. 7 b is that the enzyme detecting specificity mir-181a-5pRNA probe area in miRNA detection chip under the electrically and biologically fluorescent microscope of an embodiment cuts rear fluorescence intensity photo.
Embodiment
Mainly in conjunction with the drawings and the specific embodiments explanation is further explained to miRNA detection chip and preparation method thereof and application below.
As shown in Figure 1, the miRNA detection chip 100 of an embodiment, comprise be provided with golden film substrate 110, specific RNA probe 120 and ssDNA probe 130.
Substrate is flat glass substrate, and golden film is located at the side of substrate.
One end modified biological element of specific RNA probe 120, vitamin H is combined with fluorescent marker, the other end of specific RNA probe 120 modifies sulfydryl, and one end that specific RNA probe 120 is modified with sulfydryl is combined with golden film by Au-S key, thus is fixed on and is provided with in the substrate 100 of golden film.
One end modified biological element of ssDNA probe 130, vitamin H is combined with fluorescent marker, the other end of ssDNA probe 130 modifies sulfydryl, and one end that ssDNA probe 130 is modified with sulfydryl is combined with golden film by Au-S key, thus is fixed on and is provided with in the substrate 100 of golden film.
Concrete, the fluorescent marker that vitamin H combines can be the streptavidin that fluorescein isothiocyanate (FITC) marks, fluorescein isothiocyanate (FITC) is marked on streptavidin, vitamin H and streptavidin have good affinity, vitamin H can be combined with fluorescently-labeled Streptavidin and form vitamin H-Streptavidin-fluorescein isothiocyanate system, realizes the amplification of detection signal in follow-up testing process.
Concrete, miRNA detection chip 100 is micro-array chip.
In one embodiment, miRNA detection chip 100 is fixed with a kind of specific RNA probe 120, the specific sequence that specific RNA probe 120 designs containing with good grounds mir-29a-3p or mir-181a-5p, as shown in SEQIDNo.1 or SEQIDNo.2.
In another embodiment, miRNA detection chip 100 is fixed with two species specificity rna probes 120, specific RNA probe 120 is containing the specific sequence of with good grounds mir-29a-3p and mir-181a-5p design, and the sequence of two species specificity rna probes 120 is respectively as shown in SEQIDNo.1 and SEQIDNo.2.
In one embodiment, miRNA detection chip 100 is fixed with the ssDNA probe 130 for detecting contrast, the sequence of ssDNA probe is according to the sequences Design of specific RNA probe 120, uridylic base (U) in the sequence of the specific RNA probe 120 designed is replaced with thymine alkali bases (T), obtains the sequence of ssDNA probe.Concrete, the sequence of ssDNA probe can as shown in SEQIDNo.3.
Be appreciated that in other embodiments, the sequence of specific RNA probe 120 and ssDNA probe 130 is not limited thereto, and according to the sequence of the target miRNA that will detect, can design different specific RNA probes 120 and ssDNA probe.According to the needs detected, a kind of, two kinds of even multiple specific RNA probes 120 can be fixed in miRNA detection chip 100.
Not only containing specific RNA probe but also containing the ssDNA probe for detecting contrast in miRNA detection chip, and all with fluorescent mark material.During detection, ssDNA probe can be used as object of reference, detect the validity of miRNA detection chip on the one hand, on the other hand can as the contrast reference of specific RNA probe in detecting result, in same chip, detect to obtain experimental group and experiment contrast group, improve the reliability of detection data.
This miRNA detection chip, specific RNA probe and ssDNA probe are all combined with fluorescent marker, and specific RNA probe contains the specific sequence for detecting target miRNA.When the cDNA that miRNA reverse transcription to be measured is formed can be hybridized with this specific sequence, form miRNA/cDNA crossbred.The miRNA in miRNA/cDNA crossbred is cut again by use rnase enzyme, the fluorescent marker be combined on specific RNA probe is departed from, rear fluorescence intensity is cut by comparable chip initial fluorescent intensity and enzyme, obtain the degradation amount of the specific RNA probe on miRNA detection chip surface, thus obtain the content of miRNA to be measured.Simultaneously, the cDNA of release can be combined with the specific RNA sequence of chip surface repeatedly, then is hydrolyzed, until the RAN probe of chip surface is hydrolyzed completely, thus define a kind of amplification detection effect based on non-PCR, the high throughput testing that can realize miRNA easy and simple to handle.
The making method of above-mentioned miRNA detection chip as shown in Figure 2, comprises the steps:
S110, build specific sequence for detecting target miRNA, this specific sequence can be hybridized with the cDNA formed by target miRNA reverse transcription, this specific sequence one end modified biological element and fluorescent marker is combined on vitamin H, then modify sulfydryl at the other end, obtain specific RNA probe.
In one embodiment, target miRNA is a kind of, mir-29a-3p or mir-181a-5p, and the specific sequence of structure designs, specifically as shown in SEQIDNo.1 or SEQIDNo.2 according to mir-29a-3p or mir-181a-5p.
In another embodiment, target miRNA has two kinds, is respectively mir-29a-3p and mir-181a-5p, builds two species specificity sequences, respectively as shown in SEQIDNo.1 and SEQIDNo.2.
S120, build single-stranded DNA sequence for detecting contrast, this single-stranded DNA sequence one end modified biological element and fluorescent marker is combined on vitamin H, then modify sulfydryl at the other end, obtain ssDNA probe.
Single-stranded DNA sequence for detecting contrast can design according to specific RNA probe sequence, the uridylic base (U) built in the specific sequence detecting target miRNA is replaced with thymine alkali bases (T), obtains the sequence of ssDNA probe.Concrete, the sequence of ssDNA probe can as shown in SEQIDNo.3.
Concrete, fluorescent marker can be the streptavidin that fluorescein isothiocyanate (FITC) marks, vitamin H and streptavidin have good affinity, vitamin H can be combined with fluorescently-labeled Streptavidin and form vitamin H-Streptavidin-fluorescein isothiocyanate system, realizes the amplification of detection signal in follow-up testing process.
S130, substrate surface formed one deck gold film, obtain the substrate being provided with golden film.
In the ssDNA probe that S140, the specific RNA probe obtained by S110 and S120 the obtain substrate being provided with golden film that point sample obtains to S130 respectively, 4 DEG C of sealings are preserved, and make specific RNA probe and between ssDNA probe and the substrate being provided with golden film, the self-assembly of Au-S key occur and be fixed probe.
General, will to the substrate clean being provided with golden film before point sample.
Concrete, specific RNA probe and ssDNA probe exist as a solution, specific RNA probe and ssDNA probe are diluted to 1 μm of ol/L, and point sample is in the substrate being provided with golden film.
Concrete, the condition that sealing is preserved is that 4 DEG C of lower seals preserve 16h, and specific RNA probe and ssDNA probe are fully combined with the substrate being provided with golden film, by probe with sulfydryl and golden film between there is the self-assembly of Au-S key and probe be fixed.
S150, employing ethanol solution close white space, obtain miRNA detection chip.
Concrete, after miRNA detection chip fixes probe, soak more than two hours with the mPEG-SH ethanol solution of 1mmol/L, close the white space of chip surface, namely obtain miRNA detection chip.
Concrete, the miRNA detection chip of structure is micro-array chip.
Specific RNA probe in above-mentioned miRNA detection chip and ssDNA probe are all combined with fluorescent marker, and specific RNA probe contains the specific sequence for detecting target miRNA.When the cDNA that miRNA reverse transcription to be measured is formed can be hybridized with this specific sequence, form miRNA/cDNA crossbred.The miRNA in miRNA/cDNA crossbred is cut again by use rnase enzyme, the fluorescent marker be combined on specific RNA probe is departed from, rear fluorescence intensity is cut by comparable chip initial fluorescent intensity and enzyme, obtain the degradation amount of the specific RNA probe on miRNA detection chip surface, thus obtain the content of miRNA to be measured.Simultaneously, the cDNA of release can be combined with the specific RNA sequence of chip surface repeatedly, then is hydrolyzed, until the RAN probe of chip surface is hydrolyzed completely, thus define a kind of amplification detection effect based on non-PCR, the high throughput testing that can realize miRNA easy and simple to handle.
The detection method of a kind of miRNA as shown in Figure 3, it comprises the steps:
S210, extract miRNA to be measured, and with the miRNA to be measured extracted for template, under the effect of reversed transcriptive enzyme, reverse transcription forms the cDNA with miRNA complementation to be measured.
Concrete, extract miRNA to be measured from sample to be tested after, can adopt specificity stem ring reverse transcriptase primer that miRNA reverse transcription in sample is become cDNA.
S220, the miRNA detection chip made is placed in fluorescent microscope under carry out fluoroscopic examination, obtain initial fluorescent intensity.
Concrete, fluorescent microscope is electrically and biologically fluorescent microscope.
S230, in miRNA detection chip, add the cDNA with miRNA complementation to be measured, obtain miRNA/cDNA crossbred.
After have detected the initial fluorescence of chip, chip is taken out from fluorescent microscope, be placed on plane operations platform, add the cDNA that a certain amount of above-mentioned miRNA reverse transcription becomes.If can hybridize with the rna probe in miRNA detection chip containing target miRNA, the then cDNA be transcribed into by it in sample, form miRNA/cDNA crossbred.
S240, the miRNA added in Yeast Nucleic Acid enzymic hydrolysis miRNA/cDNA crossbred, discharge the cDNA in crossbred.
Concrete, rnase is the concentration of RNaseH, RNaseH is 60U/mL.RNaseH is a kind of endoribonuclease, can be hydrolyzed the RNA in DNA and RNA heterozygosis chain specifically, and can not phosphodiester bond in hydrolysing single or double-stranded DNA or RNA, namely can not hydrolysing single or double-stranded DNA or RNA.
Concrete, hydrolysis operation is dripped by rnase on miRNA detection chip surface, be paved with probe area, leave standstill 10 minutes, make the miRNA in rnase cutting miRNA/cDNA crossbred, discharge the cDNA in crossbred, the cDNA discharged continues again the remaining complete miRNA probe chain with chip surface and is hybridized, is hydrolyzed.Circulation hydrolysis, realizes causing a large amount of miRNA hydrolysis with a small amount of cDNA.
CDNA, rnase and fluorescent marker that S250, washing removing dissociate, carry out fluoroscopic examination under miRNA detection chip being placed in fluorescent microscope, obtain enzyme and cut rear fluorescence intensity.
RNase-freewater (not the water of qiagen rnase enzyme) is adopted to rinse chip surface, the miRNA etc. of cDNA free on removing chip, rnase, fluorescent marker and hydrolysis, carry out fluoroscopic examination under again miRNA detection chip being placed in fluorescent microscope, obtain enzyme and cut rear fluorescence intensity.
If desired two or more miRNA is detected, adopt the detection chip containing two or more specific RNA probe, after detecting a kind of miRNA by above-mentioned steps, detection chip is regenerated, chip regeneration step is the NaOH solution regeneration chip surface adopting 10mmol/L, rinse chip surface with RNase-freewater (not the water of qiagen rnase enzyme) again, detect another kind of miRNA by above-mentioned steps afterwards.
S260, by comparing initial fluorescent intensity and enzyme cuts rear fluorescence intensity, obtaining the degradation amount of the specific RNA probe on miRNA detection chip surface, thus obtaining the content of miRNA to be measured.
Cut rear fluorescence intensity whether can detect qualitatively in sample to be tested containing target miRNA by comparing initial fluorescent intensity and enzyme.
Further, by adding the cDNA of concentration known in the chips, the typical curve that cDNA concentration and initial fluorescent intensity and enzyme cut the difference of rear fluorescence intensity can be obtained.Subsequently the cDNA that the miRNA reverse transcription that sample to be tested extracts is formed is added chip, obtain the difference that the initial fluorescent intensity of sample and enzyme cut rear fluorescence intensity, by the content calculating the target cDNA that miRNA reverse transcription to be measured is formed that typical curve can be quantitative, target miRNA content in anti-release sample to be tested further.
The detection method of above-mentioned miRNA, not only containing specific RNA probe but also containing the ssDNA probe for detecting contrast in the miRNA detection chip used, and all with fluorescent mark material.During detection, ssDNA probe can be used as object of reference, detect the validity of miRNA detection chip on the one hand, on the other hand can as the contrast reference of specific RNA probe in detecting result, test experience group and experiment contrast group in same chip, improve the reliability detecting data.Cut rear fluorescence intensity by comparable chip initial fluorescent intensity and enzyme, obtain the degradation amount of the specific RNA probe on miRNA detection chip surface, thus obtain the content of miRNA to be measured.Simultaneously, the cDNA of release can be combined with the specific RNA sequence of chip surface repeatedly, then is hydrolyzed, until the RAN probe of chip surface is hydrolyzed completely, thus define a kind of amplification detection effect based on non-PCR, the high throughput testing that can realize miRNA easy and simple to handle.The detection method of this miRNA, by easy detection technique of fluorescence platform, be aided with efficient cyclophorase and cut signal amplification technique, realize the Sensitive Detection of miRNA, for the examination of disease-related miRNA express spectra provides effective means, there are stronger practical application potentiality.
It is below specific embodiment part.
In following examples, if no special instructions, the experimental technique of unreceipted actual conditions, usually conveniently condition,
Instrument: electrically and biologically fluorescent microscope (German Zeiss); Chip (Suzhou Siju Biomaterials Co., Ltd.).
Reagent: specific RNA probe and ssDNA probe (Dalian Takara company); Probes complementary cDNA (Dalian Takara company); RNaseH enzyme (Dalian Takara company).
Embodiment 1 builds miRNA detection chip
Build the miRNA detection chip containing two species specificity rna probes, can detect mir-29a-3p and mir-181a-5p, two species specificity sequences are respectively as shown in SEQIDNo.1 and SEQIDNo.2.The sequence of single stranded DNA is as shown in SEQIDNo.3.The specific sequence built one end modified biological element and the streptavidin that fluorescein isothiocyanate (FITC) marks is combined on vitamin H, then modify sulfydryl at the other end, obtain specific RNA probe and DNA probe.The specific RNA probe of the 1 μm of ol/L diluted and ssDNA probe are distinguished manual point sample surperficial in the chip gold film that clean is good, 4 DEG C seal preservation 16 hours, make between sulfhydrylation probe and golden film, the self-assembly of Au-S key occur and be fixed probe.After probe fixes, adopt the mPEG-SH ethanol solution of 1mmol/L to soak more than 2 hours, closed chip surface is the white space of stationary probe.Carry out fluoroscopic examination under chip being placed on electrically and biologically fluorescent microscope, record its fluorescence intensity, be designated as initial fluorescent intensity.
Embodiment 2 production standard curve
From microscope, take out chip, be placed on plane operations platform, add the standard cDNA that the mir-29a-3p of a series of concentration known is corresponding, leave standstill 10 minutes.Add the RNaseH enzyme solution of 60U/mL, be paved with probe area, leave standstill 10 minutes, make the miRNA in RNaseH enzyme cutting miRNA/cDNA crossbred, discharge the cDNA in crossbred, the cDNA discharged continues again the remaining complete miRNA probe chain with chip surface and is hybridized, is hydrolyzed.Circulation hydrolysis, realizes causing a large amount of miRNA hydrolysis with a small amount of cDNA.CDNA, RNaseH enzyme free with RNase-freewater washing removing and fluorescent marker, carry out fluoroscopic examination under miRNA detection chip being placed in fluorescent microscope, obtain enzyme and cut rear fluorescence intensity.The relation of cDNA concentration and Δ I (initial fluorescent intensity-enzyme cuts rear fluorescence intensity) is as shown in table 1:
Table 1:cDNA concentration and Δ I relation table
According to table 1 draw typical curve as shown in Figure 4.
Embodiment 3cDNA and ssDNA probe desmoenzyme are cut front and back fluorescence intensity and are contrasted
Detect the initial fluorescent intensity photo in ssDNA probe region in miRNA detection chip under electrically and biologically fluorescent microscope (German Zeiss), detect fluorescence intensity is 1380, initial fluorescent intensity photo as shown in Figure 5 a.The standard cDNA sample drop of 100nmol/L synthetic is added to ssDNA probe region, leaves standstill 10 minutes, itself and single stranded DNA are fully hybridized.Afterwards the RNaseH enzyme solution of 60U/mL is added drop-wise to chip surface, leaves standstill 10 minutes.Free cDNA, RNaseH enzyme and fluorescent marker is removed again with RNase-freewater washing.Carry out fluoroscopic examination under miRNA detection chip being placed in fluorescent microscope, obtain enzyme and cut rear fluorescence intensity, detect fluorescence intensity is 1300, enzyme cuts rear fluorescence intensity photo as shown in Figure 5 b.From Fig. 5 a and Fig. 5 b, initial fluorescent intensity and enzyme are cut rear fluorescence intensity and are more or less the same, and illustrate that a RNaseH enzyme specificity works for the RNA in DNA and RNA heterozygosis chain.
Embodiment 4cDNA and rna probe desmoenzyme are cut front and back fluorescence intensity and are contrasted
Detect the initial fluorescent intensity of specificity mir-29a-3pRNA probe area in miRNA detection chip under electrically and biologically fluorescent microscope (German Zeiss), detect fluorescence intensity is 1400, initial fluorescent intensity photo as shown in Figure 6 a.Standard cDNA sample drop corresponding for the mir-29a-3p of 100nmol/L synthetic is added to rna probe region, leave standstill 10 minutes, itself and rna probe are fully hybridized, obtain miRNA/cDNA crossbred, afterwards the RNaseH enzyme solution of 60U/mL is added drop-wise to chip surface, leave standstill 10 minutes, RNaseH enzyme enzyme is made to cut miRNA in miRNA/cDNA crossbred, again with the cDNA that RNase-freewater washing removing is free, RNaseH enzyme and fluorescent marker, fluoroscopic examination is carried out under miRNA detection chip being placed in fluorescent microscope, obtain enzyme and cut rear fluorescence intensity, detect fluorescence intensity is 480, enzyme cuts rear fluorescence intensity photo as shown in Figure 6 b.
Adopt the NaOH solution regeneration miRNA detection chip surface of 10mmol/L, then rinse chip surface with RNase-freewater (not the water of qiagen rnase enzyme).Detect the initial fluorescent intensity of specificity mir-181a-5pRNA probe area in miRNA detection chip under electrically and biologically fluorescent microscope (German Zeiss), detect fluorescence intensity is 1450, initial fluorescent intensity photo as shown in Figure 7a.Standard cDNA sample drop corresponding for the mir-181a-5p of 100nmol/L synthetic is added to rna probe region, leaves standstill 10 minutes, itself and rna probe are fully hybridized and obtains miRNA/cDNA crossbred.Afterwards the RNaseH enzyme solution of 60U/mL is added drop-wise to chip surface, leaves standstill 10 minutes, make RNaseH enzyme enzyme cut miRNA in miRNA/cDNA crossbred.Free cDNA, RNaseH enzyme and fluorescent marker is removed again with RNase-freewater washing, fluoroscopic examination is carried out under miRNA detection chip being placed in fluorescent microscope, obtain enzyme and cut rear fluorescence intensity, detect fluorescence intensity is 350, enzyme cuts rear fluorescence intensity photo as shown in Figure 7b.
As can be seen from Fig. 6 a, Fig. 6 b, Fig. 7 a and Fig. 7 b, the standard cDNA sample corresponding with specific RNA sequence is added in rna probe region, add RNaseH enzyme enzyme again to cut, the initial fluorescent intensity that enzyme cuts rear fluorescence intensity ratio chip reduces, because RNaseH enzyme energy specificity works for the RNA in DNA and RNA heterozygosis chain, by the miRNA hydrolysis in miRNA/cDNA crossbred.
The detection of miRNA in embodiment 5 first stream patient throat swab sample
MiRNA is extracted from first stream patient throat swab sample, adopt specificity stem ring reverse transcriptase primer that mir-29a-3p reverse transcription in sample is become cDNA, instill in the chip built, detect the initial fluorescent intensity of chip is 1212, the RNaseH enzyme solution of 60U/mL is added drop-wise to chip surface, leave standstill 10 minutes, make RNaseH enzyme enzyme cut miRNA in miRNA/cDNA crossbred.Again with free cDNA, RNaseH enzyme of RNase-freewater washing removing and fluorescent marker, carry out fluoroscopic examination under miRNA detection chip being placed in fluorescent microscope, obtaining enzyme, to cut rear fluorescence intensity be 960.Δ I (initial fluorescent intensity-enzyme cuts rear fluorescence intensity) equals 252, and according to the typical curve in Fig. 4, the cDNA content extrapolating reverse transcription one-tenth is 15nmol/L, and the anti-amount pushing away mir-29a-3pmiRNA in detection sample is 15nmol/L.
The above embodiment only have expressed one or more embodiments of the present invention, and it describes comparatively concrete and detailed, but therefore can not be interpreted as the restriction to the scope of the claims of the present invention.It should be pointed out that for the person of ordinary skill of the art, without departing from the inventive concept of the premise, can also make some distortion and improvement, these all belong to protection scope of the present invention.Therefore, the protection domain of patent of the present invention should be as the criterion with claims.
Claims (10)
1. a miRNA detection chip, is characterized in that, comprise be provided with golden film substrate, specific RNA probe and ssDNA probe;
Described specific RNA probe contains the specific sequence for detecting target miRNA, and described specific sequence can be hybridized with the cDNA formed by described target miRNA reverse transcription;
One end modified biological element of described specific RNA probe, described vitamin H is combined with fluorescent marker, the other end of described specific RNA probe modifies sulfydryl, described specific RNA probe modification have one end of sulfydryl be fixed on by Au-S key described in be provided with in the substrate of golden film;
Described ssDNA probe is for detecting contrast, one end modified biological element of described ssDNA probe, described vitamin H is combined with fluorescent marker, the other end of described ssDNA probe modifies sulfydryl, is provided with in the substrate of golden film described in one end that described ssDNA probe is modified with sulfydryl is fixed on by Au-S key.
2. miRNA detection chip according to claim 1, is characterized in that, described specific RNA probe is a kind of, and the sequence of described specific RNA probe is SEQIDNo.1 or SEQIDNo.2.
3. miRNA detection chip according to claim 1, is characterized in that, described specific RNA probe is two kinds, and the sequence of described specific RNA probe is respectively SEQIDNo.1 and SEQIDNo.2.
4. miRNA detection chip according to claim 1, is characterized in that, the sequence of described ssDNA probe is as shown in SEQIDNo.3.
5. miRNA detection chip according to claim 1, is characterized in that, described fluorescent marker is the streptavidin of marked by fluorescein isothiocyanate.
6. a making method for the miRNA detection chip according to any one of Claims 1 to 5, is characterized in that, comprise the steps:
Build the specific sequence for detecting target miRNA, described specific sequence can be hybridized with the cDNA formed by target miRNA reverse transcription, described specific sequence one end modified biological element and fluorescent marker is combined on described vitamin H, then modify sulfydryl at the other end of described specific sequence, obtain specific RNA probe;
Build single-stranded DNA sequence for detecting contrast, described single-stranded DNA sequence one end modified biological element and fluorescent marker is combined on described vitamin H, then modify sulfydryl at the other end of described single-stranded DNA sequence, obtain ssDNA probe;
Form one deck gold film at substrate surface, obtain the substrate being provided with golden film;
By described specific RNA probe and described ssDNA probe respectively point sample to described in be provided with in the substrate of golden film, 4 DEG C of sealings are preserved, and make described specific RNA probe and between described ssDNA probe and the described substrate being provided with golden film, the self-assembly of Au-S key occur and be fixed probe; And
Adopt ethanol solution to close white space, obtain described miRNA detection chip.
7. a detection method of miRNA, is characterized in that, comprises the steps:
Extract miRNA to be measured, and with the miRNA described to be measured extracted for template, under the effect of reversed transcriptive enzyme, reverse transcription forms the cDNA with described miRNA complementation to be measured;
Carry out fluoroscopic examination under miRNA detection chip such as according to any one of Claims 1 to 5 is placed in fluorescent microscope, obtain initial fluorescent intensity;
In described miRNA detection chip, add the cDNA with described miRNA complementation to be measured, obtain miRNA/cDNA crossbred;
Add the miRNA in miRNA/cDNA crossbred described in Yeast Nucleic Acid enzymic hydrolysis, discharge the cDNA in crossbred;
CDNA, rnase and fluorescent marker that washing removing is free, carry out fluoroscopic examination under described miRNA detection chip is placed in fluorescent microscope, obtain enzyme and cut rear fluorescence intensity; And
Cut rear fluorescence intensity by more described initial fluorescent intensity and described enzyme, obtain the degradation amount of the specific RNA probe on miRNA detection chip surface, thus obtain the content of miRNA to be measured.
8. the detection method of miRNA according to claim 7, it is characterized in that, describedly obtain after enzyme cuts rear fluorescence intensity, also comprise chip regeneration step, described chip regeneration step is the NaOH solution regeneration chip surface adopting 10mmol/L, then rinses chip surface with the aqueous solution of not qiagen rnase enzyme.
9. the detection method of miRNA according to claim 7, it is characterized in that, in the operation of the described miRNA added in miRNA/cDNA crossbred described in Yeast Nucleic Acid enzymic hydrolysis, described hydrolysis operation is dripped by rnase on described miRNA detection chip surface, be paved with probe area, leave standstill 10 minutes, rnase is made to cut miRNA in described miRNA/cDNA crossbred, discharge the cDNA in crossbred, the cDNA discharged continues again the remaining complete miRNA probe chain with chip surface and is hybridized, is hydrolyzed.
10. the detection method of miRNA according to claim 7, is characterized in that, described rnase is RNaseH, and the concentration of described RNaseH is 60U/mL.
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