CN1381589A - Simple and fast technique for testing mRNA chromatographic chip - Google Patents
Simple and fast technique for testing mRNA chromatographic chip Download PDFInfo
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Abstract
A simple and fast mRNA chromatographic hybrid technique for detecting chip includes such steps as arraying and fixing cDNA probes on chromatographic film, dissolving the specimen by strong modifying agent to release RNA, regulating ion intensity, adding biotin-marked second probe oligonucleotide (dT) and avidin-coated colloidal gold particle solution, chromatographic hybrid detection and analyzing signals. Its advantages are simple process and short time (less than 1 hr).
Description
The present invention relates to a kind of detection technique of simple fast m RNA chromatography detection chip, refer to that specifically cDNA probe that free mRNA is fixed captures and by the detection method that original position develops the color, comprise the making of film bar probe array, RNA rapid extraction, chromatography hybridization and signal analysis etc. in the chromatography transition process.This technology is simple fast, reliable, can be used for the laboratory study and the clinical diagnosis of gene expression profile.
Along with the progress of the Human Genome Project, the ordering of all DNA sequence is decoded substantially.The mass data that structural genomics produces is effectively used; functional genomics faces a challenging great development stage; genomics still is that protein science is all emphasized on the integral level of organ-tissue the mechanics of gene to be studied; promptly mRNA or protein product are carried out mass-producing research, understand vital movement rule in certain scope by the interaction between gene active difference and its product.Thereby quickened the process of decoding life.Wherein Chang Yong method has 2-D electrophoresis connexus spectrum analysis method, and the extremely promising method of another kind is exactly the DNA chip, and the DNA chip is that a kind of collection is microminiaturized, parallelization, and comparative studies is in the research means of one.
The DNA chip technology formally came out in nineteen ninety-five, was the integrated DNA detection technology of a kind of height.At first adopt Special Automaticization equipment that several, tens, several thousand even several ten thousand known dna oligonucleotide probes or complete sequence gene probe are arranged on slide, silicon chip or the membranous material, with all DNA compositions in the sample to be detected and chip probe hybridization, there is corresponding target DNA corresponding hybridization signal will occur then.Can accomplish once to test the gene that all genes maybe can be expressed in the detection cell or tissue in theory.Therefore this technology provides best research means to research, the research of disease development and the research of physiological and pathological of fetal development control.Main application has:
1) research of gene expression difference: with thousands of even tens thousand of cDNA/mRNA fragment point samples or be fixed in glass or the silicon core
On the sheet,, the mRNA in (growth, growth, extraneous factor etc.) tissue under the specified conditions hybridizes by being carried out randomness,
By analysis software the result is analyzed, and then analyze the gene expression profile of particular organization.
2) screening of genomic medicine: the drug molecule at random that will be in a large number produces by combinatorial chemical method by molecular designing or simulation and
Polygene in the tissue carries out the drug molecule that the chance mechanism screening has the specific combination effect, is one of pharmacogenomics
Plant novel effective ways.
3) clinical diagnosis and prognosis detect: can carry out various simultaneously with the chip that contains multiple pathogenic micro-organism gene or Disease-causing gene
This detection has improved the detection effect greatly.
4) environmental detects: the many chemical substances in the physical environment impact human and other biological existence.Example
As, can detect the something lost of human various complex disease (asthma, diabetes, hypertension etc.) simultaneously with the DNA chip method
Pass the susceptibility factor.
5) protein chip: with albumen be probe stationary on solid support thing surface, form protein chip, can be used for detecting egg
Interaction between white, and the activity that can not interact.
The DNA chip that is used to detect the mRNA expression status generally with all or part of sequence of cDNA as probe stationary in chip surface, the number of cDNA probe is decided according to purpose, generally can select tens to several thousand, detection technique is ultimate principle with the nucleic acid hybridization, elder generation's extracting RNA or mRNA during detection, RNA behind the purifying carries out reverse transcription and while mark fluorescent element again, then a large amount of cDNA probes on the target sequence of mark and the chip are hybridized, the final hybridization signal that obtains the best by scanning, collect fluorescent signal by computer, and analyze after the fluorescence intensity digitizing to each point.
But this class high integration cDNA chip technology also exists following defective and deficiency:
1. need special-purpose test set
Existing chip technology all is the detected target DNA molecule of fluorescent mark technical mark that adopts hypersensitivity, so the detection of chip just needs expensive special chip laser scanner.
2. the trace routine complexity detects consuming time oversize
Existing DNA chip detection technology just fixedly is carried out reverse hybridization with dna probe is integrated, compares with traditional hybridization technique, and trace routine is still complicated, needs to operate through the professional and technical personnel of training, generally need 2 days time just can finish at least.And the requirement of clinical diagnosis project at first is quick and easy and simple to handle, and report can be finished and provide to general inspection preferably in 1～2 hour.
3. detect the cost height
Because the production process complexity of chip, the general slide that needs to adopt special modification needs special point sample equipment and test set, and owing to the reagent costliness of PCR and mark, the detection cost of increase causes the cost of chip high simultaneously.And detect simultaneously that not related gene is not clinical needed on several thousand functions of hundreds of, what need clinically is to have the special test item that also can solve practical problems at type, such as, understand oncogene expression, understand the situation or the like in the possible mutational site of institute of thalassemic globin gene.
4. need specific nuclease free environment
Need the environment of special nuclease free carrying out RNA when operation, operator had relatively high expectations, even require full-time testing staff because RNA is very responsive to the nuclease that pollutes in the environment, operate careless slightly, the just result of influence detection.
It is simpler to the purpose of this invention is to provide a kind of trace routine, can carry out the chip method that mRNA detects fast.
In order to overcome the deficiencies in the prior art and defective, the present invention takes a kind of new bar shaped chip design and chromatography detection method to reach purpose of the present invention, by improving the extractive mode of RNA, the carrier of cDNA probe stationary and the mode of hybridization, reach the detection of quick, easy gene expression profile.
The present invention relates to the technology and the method for the diagnosis of mRNA flash chromatography, comprise probe array making, RNA extraction, chromatography hybridization and signal analysis etc.This method is simple fast, reliable, can be used for laboratory study and clinical diagnosis, and this technological step comprises:
A. with the cDNA probe contain the cyclic plasmid of complementary sequence or oligonucleotide probe array on microporous fiber membranes, make film
The strip array chip;
The cell or the sample that b. will contain mRNA dissolve with strong denaturing agent, discharge RNA;
C. regulate the ionic concn of denaturant solution;
D. the colloid gold particle that in sex change liquid, adds biotin labeled Poly (T) and avidin bag quilt;
E. the end with the film bar immerses denaturant solution, and an end links to each other with water-absorption fiber, and the chromatography hybridization sample shows signal;
F. silver-colored recovering signal is strengthened, signal analysis.
Wherein c, f are optional step.Below will describe the implementation procedure that the RNA chromatography detects in detail.
1. use the cDNA probe
CDNA as probe can be by corresponding cDNA clonal expansion, also the method that can use reverse transcription to add pcr amplification is obtained probe, yet obtain the large-scale cDNA probe, use the cDNA clone bank of same plasmid vector will come economically with identical amplimer and convenience.CDNA can be a total length, also can be the part segment, and its cDNA segment promptly may obtain normal fixed effect greater than 200bp and hybridization detects effect.In order to obtain the hybridization efficiency of best effect, take following measures:
(1) volume of increase hybridization solution because the renaturation of DNA and the complicacy of DNA and the efficient of DNA concentration and hybridization have substantial connection, can reduce the renaturation of DNA in the solution by the volume that increases hybridization solution;
(2) chromatography hybridization, because the increase of hybridization solution volume, the essential chromatography hybridization technology that adopts increases the chance that contacts of target dna and stationary probe.
(3) strong denaturing agent extracting RNA is avoided the degraded of nuclease to RNA.
2. the network of probe is three-dimensional fixing
Nitrocellulose membrane, nylon membrane, pvdf membrane etc. all are the worry films that has micropore, its aperture is generally about 0.45 μ m～10 μ m, the general film of 5 μ m of selecting is as the chromatography support membrane, at this film of microscopically is exactly a spatial fibre network, liquid can carry out freely spreading among this fibre network, when when an end of film is given the liquid diffusion pressure, liquid will spread in a certain direction, for example along the folk prescription of strip film bar to diffusion.Therefore nitrocellulose membrane and other chromatographic film are spatial network immobilization carrier, are again the good material of chromatography, are again the maximum support carriers of cDNA probe stationary simultaneously.
The normal technique for fixing of cDNA probe on film relates to the covalently bound of the point sample of cDNA probe and probe and film.Can select cationic membrane for use, as amido modified nylon membrane, nitrocellulose membrane etc., can employ the three-dimensional probe stationary technology stationary probe of solid chip surface, also can adopt the method for spray line or specking that probe and contrast probe are sprayed on the nitrocellulose membrane or nylon membrane of 5mm left and right sides width, form linear array or point-like array.Through uviolizing, impel the covalent cross-linking of probe and film after the drying.
3. strong denaturing agent extracting RNA
Compare with traditional method, the present invention has a lot of advantages.The present invention directly adopts powerful denaturing agent sex change with cell sample, makes it to discharge nucleic acid, suppresses the degraded of RNA enzyme to RNA simultaneously, and makes operation more easy.Do not need the separation and the purification step of nucleic acid.Denaturing agent is a kind of RNA or DNA that can dissolve in the biological specimen, and protein is discharged from RNA and dna molecular, thereby RNA and dna molecular are fully exposed.These denaturing agents destroy RNA, DNA and proteinic secondary structure by intervening intermolecular faint van derWaal gravitation in the biological specimen.These denaturing agents almost can dissolve all biomolecules such as protein and nucleic acid.
The notion of denaturing agent is to dissolve all biomolecules, produces a kind of clear solution clearly, and the 1ml denaturant solution is the cell or tissue of solubilized 1mg at least, and therefore not all salt all can become denaturing agent.Special denaturing agent comprises trifluoroacetate, Trichloroacetate, perchlorate, NaI, guanidine thiocyanate and potassium sulfocyanate etc.The concentration of denaturing agent is different according to institute's dissolved sample.Typical concn is 5M.
4. chromatography hybridization technology
The principle design that the dna probe that the present invention mainly is fixed in the chromatography transition process according to the dissociative DNA molecule is captured.The cDNA probe array is fixed in the middle part of membrane carrier.During detection, the colloid gold reagent of biotin labeled poly (dT) fragment and avidin bag quilt is mixed with the isopyknic 6 * SSC of the strong denaturing agent solvent of RNA, and be added in the RNA denaturant solution, mix, its objective is that the ionic strength of adjusting hybridization solution is done dilution to reduce the damage to avidin to strong denaturing agent simultaneously.After Radioactive colloidal gold of RNA extracted solution and vitamin H-poly (dT) and avidin bag quilt etc. mixes, one end of film is contacted with this mixing solutions, the other end links to each other with water-absorption fiber, because chromatography effect, moving phase drives sample and launches along membrane carrier, target RNA-poly (dT)-biotin-avidin-colloidal gold composite can be captured by the respective fixation probe, and presents positive detection signal (red Radioactive colloidal gold) in corresponding band position, reaches the purpose that sharp separation is identified.Concrete grammar is as follows:
1) adopt film such as nitrocellulose filter, nylon membrane or the pvdf membrane that can allow liquid freely spread solid as the cDNA probe
Fixed chromatography carrier film, stationary probe are arranged in the central surveyed area of carrier film, form probe array;
2) strong denaturing agent extracts RNA, and (cumulative volume～1ml), 75 ℃ were heated 5 minutes in one times of 6 * SSC dilution;
3) colloid gold reagent with biotin labeled poly (dT) and avidin bag quilt mixes, and is added in the RNA solution of sex change;
4) end that will contain the carrier film of probe array is dipped in the DNA/ colloidal gold solution, and liquid launches along carrier film, film in addition
One end links to each other with water-absorption fiber, to guarantee chromatography process continuity.When the target material and the stationary probe that are marked with tracer
During pairing, just be captured fixingly, present the positive detection signal in corresponding band position;
5) reinforcement of signal, detection and analysis.The present invention compares with existing detection technique, has the following advantages:
The invention provides and can be used for that sample is prepared and the thiocyanate salt solution of hybridization, can make that sample prepares, detects that supervisor is accelerated, simple to operate, economy, multi-usage and automatization provide more advanced experimental implementation program.
1. accuracy: hybridizing in strong denaturant solution, its efficient can reach 100%, under the excessive situation of probe,
The amount of target RNA and the hybridization of probe are linear.When number of probes was low, the hybridization of target nucleic acid and probe still was
Linear relationship.
2. susceptibility: can measure the complementary nucleic acid of 30fg, this detection level can satisfy numerous detection requirements.
3. speed: no matter target RNA quantity what, all can in 15 minutes, finish DNA-RNA hybridization.The more important thing is,
So fast and under the condition of susceptibility, still can obtain so high detection speed.
4. simple: the present invention's operation only needed for three steps: 1) strong denaturant solution is added to sample or tissue, 1 minute; 2) will
The Radioactive colloidal gold of biotin labeled poly (dT) probe and avidin bag quilt and corresponding SSC diluent are added, and incubate
Educated 5 minutes; 3) chromatography hybridization analysis.The simple personnel operation that was not subjected to any training that must enough allow of this program.
The environment that nuclease is arranged is to the also few of influence of this schedule of operation.
5. economical: the expense of strong denaturing agent extracting program is all lower than all known programs.The strong denaturing agent of applied by hand
The RNA that extractive method is set up detects, and its consumption is about 2 yuan.Lower after the automatization.
6. multi-usage: clinical sample and research department's sample standard deviation can be with this method diagnosis, and for example stool, blood, urine etc. all are high
The degree miscellaneous, this technology is all effective to these samples.And do not reduce its accuracy, susceptibility, speed and economy
Indexs such as property.
7. automatization: the simplification that is better than operating is made a gesture of measuring automatization and detected becomes possibility
The present invention can be applicable to the genetic expression model and detects and analyze.
Genetic expression model (gene expression profile) is that the mRNA of genes involved in the phalangeal cell expresses the moment situation, has reflected the information of cellular activity at that time.Analysis of cells genetic expression model can know which kind of state cell is in.The present invention can detect the genetic expression spectrum detection method of 10～100 genes simultaneously, makes detection very simple, quick, can finish whole testing processes being shorter than in time of 1 hour.
Gene expression profile bar shaped chip detection principle: animal class cell mRNA all has Poly (A) sequence at 3 ' end, this sequence can be used as the recognition sequence of general probe hybridization, general probe is Poly (dT), 5 ' is marked with coloring matters such as Radioactive colloidal gold or ferritin, when mixing, just hold multiple the combination with the Poly (A) of mRNA with mRNA solution.Can be during at chromatography when this mixture along with the diffusion of liquid on film, mRNA moves to the other end.When mRNA-Poly (dT) hybridization complex in transition process be fixed on cDNA probe on the film when meeting, the cDNA probe that just is fixed is captured, original position is formed with chrominance signal.Not captive mRNA molecule continues reach, till running into the molecule that can capture.Major technology of the present invention is made and operation
The present invention relates to the technology and the method for the diagnosis of mRNA flash chromatography.This method is simple fast, reliable, can be used for laboratory study and clinical diagnosis.When being used for the mRNA detection of expression, this technological step comprises:
1) with the cDNA probe contain the cyclic plasmid of complementary sequence or oligonucleotide probe array on microporous fiber membranes, make
Film strip array chip;
2) cell or the sample that will contain mRNA dissolves with strong denaturing agent, discharges RNA;
3) ionic concn of adjusting denaturant solution;
4) colloid gold particle of adding biotin labeled Poly (T) and avidin bag quilt in sex change liquid;
5) end with the film bar immerses denaturant solution, and an end links to each other with water-absorption fiber, and the chromatography hybridization sample shows signal;
6) silver-colored recovering signal is strengthened, signal analysis.Below in detail substep describe implementation procedure 1. that the RNA chromatography detects with the cDNA probe contain the cyclic plasmid of complementary sequence or oligonucleotide probe array on microporous fiber membranes, make the film bar
Array chip
The a.cDNA probe design
The kind and the quantity of cDNA probe are mainly decided as requested, because the limitation of length of film, the number of probes of selection should not be above 200.
CDNA as probe can be by corresponding cDNA clonal expansion, also the method that can use reverse transcription to add pcr amplification is obtained probe, yet obtain the large-scale cDNA probe, use the cDNA clone bank of same plasmid vector will come economically with identical amplimer and convenience.CDNA can be a total length, also can be the part segment, and its cDNA segment promptly may obtain normal fixed effect greater than 200bp and hybridization detects effect.In order to obtain the hybridization efficiency of best effect.
B. probe point sample
The wired sample of the arrangement mode of probe is arranged or sample application array.Line sample homotaxis is in bar code, and the probe of sex change is arranged in film bar surface in order.Adopt the spray line method that probe (no more than 50) and contrast probe are sprayed on the 5mm-10mm wide nitrocellulose membrane or nylon membrane during making.The making of sample application array is mainly by means of the specimen needle of three-dimensional robot and special facture, the cDNA probe points of sex change is arrived the corresponding position of film, density is not higher than every square centimeter 200 point, and density is mainly according to the mould material that is adopted, and fine and close material can adopt higher density.
C. probe stationary
Film bar behind spray line or the point sample carries out crosslinked with ultraviolet ray (254nm) after drying at room temperature.Crosslinked energy is generally 120,000 μ j/cm
2Shone 20 seconds, and also can adjust irradiation dose as required.Another kind method is to add heat setting, and 80 ℃ of bakings reached the crosslinked purpose of DNA in 2 hours too.
D. the selection of film
The film of stationary probe mainly is a nitrocellulose membrane, as commodity BA85 (Schleicher and Schull) and HAWP (Millipore), nylon membrane such as Biodyne (trademark of Pall), Gensscreen (New England Nuclear) and Zetapore or Zetaprobe (AMF Curio).The aperture of film is generally at 5～10 μ m.2. the cell or the sample that will contain mRNA dissolve with strong denaturing agent, discharge RNA
According to following operation can be optionally with from addition extracting of the mRNA of complete blood cell, wherein strong denaturing agent is an example to add thiocyanate-GuSCN extracting.Since other operations are main in the characteristic of mRNA, in order to increase the effect that mRNA extracts.Below described respectively:
A. biological specimen is prepared
Can adopt multiple ordinary method cell in the past, carrying out the mRNA extraction is to want SC, in case the RNA degraded.Generally, sample places on ice, wears gloves, uses Cyclohexamide and adds nucleic acid inhibitor etc.Cyclohexamide (cyclohexamide) is degraded to reduce at a kind of natural structure state by keeping mRNA, nucleic acid inhibitor oxygen alum ribonucleoside (vanadylribonucleosides) suppresses the RNA enzyme, do not influence molecular hybridization, but it suppresses reverse transcription and translation.Add with 0.5mM aurin tricarboxylic acid (aurin tricarboxylic acid) (Sigma Chemicals) add 1mM Hydroxystilbamidine (hydroxystilbamidine isothamine) (Merrell) or 1U/ml RNAsin (Promega Biotech) reverse transcription or translation are not had restraining effect, but its ability that suppresses nuclease is not as oxygen alum ribonucleoside.
If sample is mononuclearcell or medullary cell, can adopt discontinuous Ficoll Hypaque density gradient centrifugation isolated cell, solid tissue can directly use thiocyanate leach solution.
B. add stain remover and thiocyanate-
The addition sequence of stain remover and thiocyanate-is not strict, earlier or after add or add all together and have not big harm.Stain remover can dissolved cell, and helps arrestin matter and the coprecipitation phenomena of DNA when chromatography.If the mRNA sample contains abundant protein or DNA, will be to add stain remover, suitable stain remover has Bu Lijie (Brij) series or tween Tween series.Weak anionic stain remover sarcosyl (sodium lauryl sarcosinate) and sodium deoxycholate (sodiumdesoxycholate) effect are also good.For example, for smudge cells, the 10%Brij 35 (Sigma) that can add 1/20 volume mixes with sample, adds 10% sodium deoxycholate of 1/20 volume then, mixes once more.
After stain remover adds, add the super-saturated thiocyanate salt solution (containing 2% beta-mercaptoethanol, 10mM Tris-HClpH 7.0,1mM EDTA) of 1 volume, make cell can obtain saturated thiocyanate-.
Strong salts solution is easy to preparation.NaI for example, over-saturation NaI approximately is that 2.5g NaI is dissolved in 75 ℃ of hot water of 1ml, room temperature storage is with before being heated to 75 ℃.Add 1 volume over-saturation NaI during use in sample, form saturated NaI extract, should see that sample solution should be clarifying this moment.The preparation method of other denaturing agent salt similarly.
C. the preparation of thiocyanate-denaturing agent
Guanidine thiocyanate 4M
Tris-HCl(pH?7.1)????10mM
Trisodium Citrate (pH 7.1) 25mM
EDTA????????????????1mM
The ionic concn that adds 3. adjusting denaturant solution before beta-mercaptoethanol 2% uses
The purpose that adds thinner mainly is the concentration of dilution denaturing agent, reduces denaturing agent to avidin-vitamin H bonded influence, adjusts the ionic strength of liquid simultaneously, make it more appropriate to hybridization.The thinner composition is: 6 * SSC, 10mM Tris-HCl (pH7.4), 1mM EDTA, 0.25%SDS.During use isopyknic thinner is added in the extractive RNA solution of denaturing agent.
70 ℃ of heat denatured 5 minutes make the abundant sex change of RNA.4. the colloid gold particle that in sex change liquid, adds biotin labeled Poly (T) and avidin bag quilt
In above-mentioned mixing liquid, add 50pmol biotin labeling Oligo (dT)
20-36, the colloid gold reagent that adds the 10OD avidin bag quilt of 100 μ l mixes, and is used for the chromatography hybridization of step 5.
Second kind of marking method is can form the stable principle design that is connected according to gold and sulfydryl.When using Radioactive colloidal gold, because Radioactive colloidal gold can form stabilized complex with sulfhydryl compound as tracer agent.Poly (dT) second probe of using marking sulfhydryl can reduce the cost of probe mark, and makes operation more easy.
Because the metallic character of gold, mercapto groups can form stable absorption with gold surface, does not need special covalent attachment, therefore makes the mark of gold grain more simple.Purifying behind the mark can adopt the method for centrifugation, the general precipitation of removing the accumulative gold grain earlier with low-speed centrifugal, and then ultracentrifugation, the probe of mark is present among the precipitation, several times centrifugal repeatedly, will be deposited at last in a small amount of 10mM PBS damping fluid (pH7.0).5. the end with the film bar immerses denaturant solution, and an end links to each other with water-absorption fiber, and the chromatography hybridization sample shows signal
Animal class cell mRNA all has Poly (A) sequence at 3 ' end, this sequence can be used as the recognition sequence of general probe hybridization, and general probe is Poly (dT), and 5 ' is marked with coloring matters such as Radioactive colloidal gold or ferritin, when mixing, just hold multiple the combination with the Poly (A) of mRNA with mRNA solution.Can be during at chromatography when this mixture along with the diffusion of liquid on film, mRNA moves up.When mRNA-Poly (dT) hybridization complex in transition process be fixed on cDNA probe on the film when meeting, the probe that just is fixed is captured, original position is formed with chrominance signal.Not captive mRNA molecule continues reach, till running into the molecule that can capture.
During detection, mRNA after sex change or RNA solution mix with the colloidal gold solution of poly (dT) and avidin bag quilt, and the centrifugation insoluble substance is noted the homogeneity of sample in the hole.Same end with film bar chip is dipped in the mixed solution then, the other end links to each other with water-absorption fiber, allow liquid spread to the other end chromatography along nylon membrane, drive the mRNA migration in the sample simultaneously, when mRNA arrived corresponding position, the probe that just is fixed was captured, general about needs 15 minutes to 30 minutes, through 15 minutes chromatography, the flush away dissociant was then by the position of visual inspection band and having or not of signal again.The quality of chip hybridization can be controlled by built-in contrast.
Under the room temperature condition, 3～6.5M GuSCN is effective equally, and hybridization can be carried out 5 minutes, also can reach a few hours.Schedule of operation is extraordinary simple.Uses such as this technology also can conjugated protein enzyme, stain remover, organic solvent, reductive agent, because biological specimen amplifying nucleic acid complexity, what have may not directly contact thiocyanate salt solution.6. silver-colored recovering signal is strengthened, signal analysis.
Can the naked eyes direct viewing contrast, what be more suitable for is that the signal that the mark colloid gold particle adds silver reduction reinforcement can be obtained by general planar optics scanner scanning, single pass can obtain detected result more than 50 parts simultaneously, automatically analyze and report and printing by image analysis software again by the image that scanning is obtained, help analyzing automatically and clinical large-scale use.
MRNA hybridization is provided with internal reference can provide quantitative reference.Measured tube and reference pipe are set during hybridization, and the result is with the ratio decision of the signal of two pipes.Good is that the amount expressed under various conditions of known mRNA is as reference with reference to pipe.Another reference is the amount of the poly (A) of total mRNA.Biotin labeled poly (dT) detects the amount of poly (A), as the reference point of other mRNA.
Parallel feminine gender and positive control all are essential for quantification of mrna or RNA, and these contrasts have the probe identical with detected sample.
The making and the detection of embodiment 1 hemopoietic stem cell part-structure protein gene expression spectrum bar shaped chip
1.cDNA the selection of probe and making
With the gene cDNA of hemopoietic stem cell part-structure albumen and some small proteins as probe stationary in the carrier film surface, gene is as shown in table 1.The cDNA of these genes is all come out by the clone, and some is a full-length gene, and some is the portion gene sequence of Poly (A) end.These gene clones can be increased it with general primer in plasmid, and the employed clone of this example is from Nanfang Research Centre, State Human Gene Group, and also there are supply in American I .M.A.G.E and InCyte company.
Table 1 part hemopoietic stem cell structural protein gene
GeneBank No expressed proteins title
X63432?????????ACTB?mRNA?for?mutant?beta-actin(beta′-actin)
U03269?????????actin?capping?protein?alpha?subunit(CapZ)
U12026?????????actin-regulatory?protein?Cap-G(CAPG)
K00558?????????alpha-tubulin
X00351?????????beta-actin
V00599?????????beta-tubulin.
M80563?????????CAPL?protein
U03851?????????capping?protein?alpha
U56637?????????capping?protein?alpha?subunit?isoform?1
X84075?????????cardiac?myosin?binding?protein-C
V00478?????????cytoplasmic?actin
X04098?????????cytoskeletal?gamma-actin
X04588?????????cytoskeletal?tropomyosin?TM30(nm)
X73882?????????E-MAP-115
U62962?????????Int-6
L25941?????????integral?nuclear?envelope?inner?membrane?protein(LBR)
M63483?????????maior?nuclear?matrix?protein
M27937?????????male-enhanced?antigen?mRNA(Mea)
U38291?????????microtubule-associated?protein?1a(MAP1A)
Y00764?????????mitochondrial?hinge?protein
M22382?????????mitochondrial?matrix?protein?P1
M69066?????????moesin
M31211?????????myosin?light?chain?1?slow?a(MLC1sa)
M31212?????????myosin?light?chain?3?non-muscle(MLC3nm)
U26162?????????myosin?regulatory?light?chain
X54304?????????myosin?regulatory?light?chain
J00312?????????non-muscle(fibroblast)tropomyosin?gene
L22343?????????nuclear?phosphoprotein
M60858?????????nucleolin
X75252?????????phosphatidylethanolamine?binding?protein
J03191?????????profilin
U61734?????????protein?trafficking?protein(S31iii125)
L03785?????????regulatory?myosin?light?chain(MYL5)
Y00282?????????ribophorin?II
U51586?????????siah?binding?protein?1(SiahBP1)
S65762?????????SPTBN1＝beta-fodrin
M25077?????????SS-A/Ro?ribonucleoprotein?autoantigen?60?kd
U26648?????????syntaxin?5
U77942?????????syntaxin?7
M25246?????????vimentin
2.cDNA probe amplification is synthetic, purifying
Use the PCR test kit of Qiagen or Promega company, according to the process specifications selected cDNA clone that increases, in the reaction process, use the appended universal PC R primer of plasmid as universal PC R synthetic primer, plasmid cDNA clone is as template, the extension time needed 2 minutes or more than, 30 circulations, product is after the electrophoresis proof, and directly alcohol (75%) precipitation gets final product.Sedimentary DNA can be directly used in behind 20 μ l, 3 * SSC solution dissolving and high-temperature denatured (98 ℃ 5 minutes, frozen water cooling immediately subsequently) and spray line or line.
3. spray line or line
Adopt spray line method (BioDot) that selected probe and contrast cDNA probe (Actin and Microglubin and DNA of plants) are sprayed on formation band array on the wide nylon membrane of 5mm～10mm, perhaps use some model machine (Cartesian) with the specified location of cDNA probe points, form the point-like array in film.Behind spray sample or the point sample, drying at room temperature, baking 2 hours in 80 ℃ of vacuum ovens is so that cDNA probe and nitrocellulose membrane or nylon membrane form covalent cross-linking.Perhaps, the film bar behind spray line or the point sample carries out crosslinked with crosslinked stove ultraviolet ray (254nm) after drying at room temperature.Crosslinked energy is generally 120,000 μ j/cm
2Shone 20 seconds, and also can adjust irradiation dose as required.
4. second probe mark
The length of Poly (dT) second probe can be chosen in 15～36 bases, and this example is selected 36 base length.5 ' end is answered the mark vitamin H, and has the connecting arm of 6 charcoal atoms between oligonucleotide and vitamin H.And the colloid gold particle (40nm) of use avidin bag quilt is as colouring reagents.
5. press mold and cutting
Above-mentioned nitrocellulose membrane or the nylon membrane that sprayed stationary probe passed through compound press mold machine, be compounded on the PVC sheet plastic, to increase the toughness and the workability of tunica fibrosa.Adopt cutting machine that compound good diaphragm is cut into required specification and shape (it is wide to be generally 2.5mm～5mm, about 50mm～100mm grows), single part with detection reagent film bar.With single minute reagent membrane bar and siccative pack into aluminium film pouch and sealing.Or single branch is detected the film bar be assemblied in the small plastic box, be equipped with corresponding chromatographic solution pouch and water-absorption fiber, make complete proofing unit, then proofing unit is placed in the dry bag.
6. biological specimen is prepared and the RNA extracting
If sample is hemocyte or medullary cell, can adopt discontinuous Ficoll Hypaque density gradient centrifugation isolated cell.Carrying out the mRNA extraction is to want SC, in case the RNA degraded.Generally, sample places on ice, wears gloves, uses Cyclohexamide and adds nucleic acid inhibitor etc.Cyclohexamide (cyclohexamide) is degraded to reduce at a kind of natural structure state by keeping mRNA, and nucleic acid inhibitor oxygen alum ribonucleoside (vanadyl ribonucleosides) suppresses the RNA enzyme.All solution all need be handled through DEPC.If the suspension cell of cultivating, directly centrifuging and taking cell precipitation.For smudge cells better, the 10%Brij 35 (Sigma) that can add 1/20 volume mixes with sample.Add guanidine thiocyanate solution (guanidine thiocyanate, 4M then; Tris-HCl (pH 7.1), 10mM; Trisodium Citrate (pH 7.1), 25mM; EDTA, 1mM; Beta-mercaptoethanol, 2% uses preceding the adding) direct extracting RNA.Generally speaking,＜10
7Cell can add 1ml guanidine thiocyanate denaturant solution, can be directly used in chromatography hybridization after the guanidine thiocyanate solution process ion adjustment after the extracting and detect.
7. adding detection reagent
The purpose that adds thinner mainly is the concentration of dilution denaturing agent, reduces denaturing agent to the influence of avidin one vitamin H bonded, adjusts the ionic strength of liquid simultaneously, make it more appropriate to hybridization.The thinner composition is: 6 * SSC, 10mM Tris-HCl (pH7.4), 1mM EDTA, 0.25%SDS.During use isopyknic thinner is added in the extractive RNA solution of denaturing agent.
70 ℃ of heat denatured 5 minutes make the abundant sex change of RNA.
In above-mentioned mixing liquid, add 50pmol biotin labeling Oligo (dT)
36, the colloid gold reagent that adds the 10OD avidin bag quilt of 100 μ l mixes, and is used for chromatography hybridization.
8. chromatography hybridization detects
During detection, one end (terminal 2mm) of film bar chip is dipped in the chromatographic solution (5 * SSC solution), the other end links to each other with water-absorption fiber, allows liquid spread to the other end chromatography along nylon membrane, drives the mRNA migration in the sample simultaneously, when mRNA arrives corresponding position, just the probe that is fixed is captured, and general about needs 15 minutes to 30 minutes are again through 15 minutes chromatography, the flush away dissociant is then by the position of visual inspection band and having or not of signal.The quality of chip hybridization can be controlled by built-in contrast.
9. signal obtains and analyzes
Can the naked eyes direct viewing contrast, what be more suitable for is that the signal that the mark colloid gold particle adds silver reduction reinforcement can be obtained by general planar optics scanner scanning, single pass can obtain detected result more than 50 parts simultaneously, automatically analyze and report and printing by image analysis software again by the image that scanning is obtained, help analyzing automatically and clinical large-scale use.
MRNA hybridization is provided with internal reference can provide quantitative reference.Measured tube and reference pipe are set during hybridization, and the result is with the ratio decision of the signal of two pipes.Good is that the amount expressed under various conditions of known mRNA is as reference with reference to pipe.Another reference is the amount of the poly (A) of total mRNA.Biotin labeled poly (dT) detects the amount of poly (A), as the reference point of other mRNA.
Parallel feminine gender and positive control all are essential for quantification of mrna or RNA, and these contrasts have the probe identical with detected sample.The quantitative chromatography of embodiment 2. leukemia cell's T cell differentiation antigen mRNA detects
T cell differentiation antigen also claims leukocyte differentiation antigen, is a kind of special sign thing that white corpuscle is expressed in cell surface, wherein much can reflect the state of process, reflection leukocyte activation or the inactivation of white corpuscle differentiation.The leukocyte surface molecule detects with antileukocytic antibody usually.The T cell differentiation antigen antibody of using various combination just can identify leukocytic hypotype, as B cell, helper cell, and cytotoxic T cell and natural killer cell etc., these cells are all inequality to the reaction and the resistance of medicine.
Leukemia is the increment of a kind of myeloid element clone property, and cell often rests on a certain stage of cytodifferentiation and the aplasia maturation, thereby can have certain special sign.Leukemia can betide any one series of hematopoietic cell, itself also has this serial distinctive mark.For acute leukemia, leukocytic immunophenotyping can be identified series and the sophisticated degree that the leukemia cell is taken place.Yet up to the present, also do not have to find, so leukemia cell's evaluation is exactly the combination evaluation that different T cell differentiation antigens are expressed usually for all special mark of any leukemia.
1. probe is selected
Up to the present, the antigen of having found with the serial anti-blood cell of naming of CD has reached numbering 166, adds various hypotypes, in fact near 190.These T cell differentiation antigen materials all are a certain specific moleculars of identification blood cell, and the overwhelming majority is protein or glycoprotein, and minority is mucopolysaccharide (a 5-6 kind).In order to reach the practicality of clinical diagnosis, the employed CD probe of this diagnosis film bar is 38, all derives from cDNA clone (plasmid), through universal PC R primer amplification gained.But these probe letters cover the diagnosis of all type of leukemia, and these probes are selected as table 2.
The CD numbering title of the selected probe of table 2.
| Numbering | Title | Function |
| ????1 | ?CD1 | ?HTA1 |
| ????2 | ?CD2 | ?E-rosette?receptor |
| ????3 | ?CD3 | ?immunoglobulin?supergene?family |
| ????4 | ?CD4 | ?L3T4 |
| ????5 | ?CD5 | ?Leu-1 |
| ????6 | ?CD7 | ?gp40 |
| ????7 | ?CD8 | ?T?cell?co-receptor |
| ????8 | ?CD10 | ?common?acute?Iymphoblastic?leukemia?antigen |
| ????9 | ?CD11c | ?AlphaL?integrin?chain |
| ????10 | ?CD13 | ?Aminopeptidase?N |
| ????11 | ?CD14 | ?LPS?receptor |
| ????12 | ?CD16 | ?FCRIIIA |
| ????13 | ?CD19 | ?B4 |
| ????14 | ?CD20 | ?B1 |
| ????15 | ?CD22 | ?Lyb8 |
| ????16 | ?CD23 | ?Leu-20，Low?affinity?IgE?receptor |
| ????17 | ?CD25 | ?IL-2?receptor?alpha?chain |
| ????18 | ?CD33 | ?gp67 |
| ????19 | ?CD34 | ?gp105-120 |
| ????20 | ?CD38 | ?ADP-ribosyl?cyclase/cyclic?ADP-ribose?hydrolase |
| ????21 | ?CD41 | ?alpha?IIb?integrin?chain |
| ????22 | ?CD56 | ?neural?cell?adhesion?molecule |
| ????23 | ?CD57 | ?Leu-7 |
| ????24 | ?CD61 | ?beta?3?integrin?chain |
| ????25 | ?Cmu | |
| ????26 | ?DR | ?DR?antigen |
| ????27 | ?HIV | |
| ????28 | ?Kappa | ?Kappa?light?chain |
| ????29 | ?Lambda | ?Lambda?light?chain |
| ????30 | ?MPO | |
| ????31 | ?NSE | |
| ????32 | ?sCD3 | |
| ????33 | ?Sig | |
| ????34 | ?TCR | |
| ????35 | ?MPO | |
| ????36 | ?TdT | |
| ????37 | ?Plasma?cell ?marker | |
| ????38 | ?β-Tubulin |
2.cDNA probe amplification is synthetic, purifying
Use the PCR test kit of Qiagen or Promega company, according to the process specifications selected cDNA clone that increases, in the reaction process, use the appended universal PC R primer of plasmid as universal PC R synthetic primer, plasmid cDNA clone is as template, the extension time needed 2 minutes or more than, 30 circulations, product is after the electrophoresis proof, and directly alcohol (75%) precipitation gets final product.Sedimentary DNA can be directly used in behind 20 μ l, 3 * SSC solution dissolving and high-temperature denatured (98 ℃ 5 minutes, frozen water cooling immediately subsequently) and spray line or line.
3. spray line or line are referring to the 3rd step of example 1.
4. second probe mark
The length of Poly (dT) second probe can be chosen in 15～36 bases, and this example is selected 36 base length.5 ' end is answered the mark vitamin H, and has the connecting arm of 6 charcoal atoms between oligonucleotide and vitamin H.And the colloid gold particle (40nm) of use avidin bag quilt is as colouring reagents.
5. press mold goes on foot referring to example 1 the 4th with cutting.
6. blood sample is handled
Collect the 15ml peripheral blood, anticoagulant heparin (50U/ml), with the 2 times of dilutions of Hanks liquid that are added with 50 μ g/ml Cyclohexamides and 10mM nucleic acid inhibitor oxygen alum ribonucleoside, with 12000g density gradient centrifugation on the cell suspension Ficoll-Hypaque 20 minutes, collect the mononuclearcell layer on Ficoll surface, wash cell 2 times with above-mentioned Hanks liquid, and cell counting.Cell concn is adjusted to 10
7/ ml adds 10%Brij-35 to ultimate density 0.5%, mixes, and adds equivalent over-saturation denaturing agent guanidine thiocyanate solution then, makes cytolysis, the solution clear.Add isopyknic 6 * SSC at last with the dilution denaturing agent, the ionic strength of regulating liquid simultaneously makes it to be suitable for hybridization, reduces denaturing agent simultaneously to avidin-vitamin H bonded influence.The thinner composition is: 6 * SSC, 10mM Tris-HCl (pH 7.4), 1mM EDTA, 0.25%SDS.During use isopyknic thinner is added in the extractive RNA solution of denaturing agent.
7.RNA sex change
70 ℃ of heat denatured of RNA solution 5 minutes with after the dilution make the abundant sex change of RNA.
5 ' the biotin labeled Poly (T)
30Provide by service provider is synthetic, with distilled water diluting to 100 μ M.In above-mentioned mixing liquid, add 50pmol biotin labeling Oligo (dT)
36, the colloid gold reagent that adds the 10OD avidin bag quilt of 100 μ l mixes, and is used for chromatography hybridization.
The avidin bag can directly be bought the colloidal gold solution (magnificent company) of commercial avidin bag quilt by Radioactive colloidal gold, also can make by oneself.
8. chromatography hybridization detects
During detection, one end (terminal 2mm) of film bar chip is dipped in the chromatographic solution (5 * SSC solution), the other end links to each other with water-absorption fiber, allows liquid spread to the other end chromatography along nylon membrane, drives the mRNA migration in the sample simultaneously, when mRNA arrives corresponding position, just the probe that is fixed is captured, and general about needs 15 minutes to 30 minutes are again through 15 minutes chromatography, the flush away dissociant is then by the position of visual inspection band and having or not of signal.The quality of chip hybridization can be controlled by built-in contrast.
9. signal obtained and analyzes referring to the 9th step of example 1.
Claims (7)
1. the present invention relates to the technology and the method for mRNA flash chromatography chip, can be used for the research of gene expression profile and the discriminating of gene active state, this technological step comprises:
A) with cDNA probe or oligonucleotide probe array on microporous fiber membranes, make film strip array chip;
B) cell or the sample that will contain mRNA dissolves with strong denaturing agent, discharges RNA;
C) ionic concn of dilution and adjusting denaturant solution, 75 ℃ of sex change RNA that heat;
D) add the colloid gold particle solution of biotin labeled Poly (T) and avidin bag quilt;
E) end with film bar chip immerses denaturant solution, and the other end links to each other with water-absorption fiber, and the chromatography hybridization sample shows letter
Number;
F) silver-colored recovering signal is strengthened, signal analysis.
2. according to the described film strip array of claim 1 chip, but it is characterized in that arranging and be fixed on cDNA probe or synthetic oligonucleotide probe on the microporous membrane of chromatography by the mode of setting-out or point sample; The material of film be nitrocellulose filter, pvdf membrane, nylon membrane and with these films be base growth come have positively charged ion or an anionic film.
3. according to the described film strip array of claim 1 chip, it is characterized in that chip is bar shaped, cDNA probe or oligonucleotide probe array are fixed in the region intermediate of film bar, and array can be arranged or dot-matrix array for the barcode sample.
4. discharge RNA according to the described strong denaturing agent of claim 1, it is characterized in that directly adopting powerful denaturing agent denaturing treatment cell or tissue sample, make it to discharge nucleic acid, suppress of the degraded of RNA enzyme simultaneously to RNA, denaturing agent comprises guanidine thiocyanate, guanidinium isothiocyanate, potassium sulfocyanate, trifluoroacetate, Trichloroacetate, perchlorate, sodium iodide etc., the concentration of denaturing agent is different according to institute's dissolved sample, and typical concn is 3～6M.
5. according to described RNA denaturing agent dilution of claim 1 and RNA sex change, it is characterized in that, colloid gold reagent with biotin labeled poly (dT) fragment and avidin bag quilt during detection mixes with the isopyknic 6 * SSC of the strong denaturing agent solvent of RNA, and be added in the RNA denaturant solution, mix, its objective is that the ionic strength of adjusting hybridization solution is done dilution to reduce the damage to avidin to strong denaturing agent simultaneously.
6. according to the described chromatography hybridization technology of claim 1, it is characterized in that after Radioactive colloidal gold of RNA extracted solution and vitamin H-poly (dT) and avidin bag quilt etc. mixes, one end of film is immersed mixing solutions, the other end links to each other with water-absorption fiber, because chromatography effect, moving phase drives sample and launches along membrane carrier, target RNA-poly (dT)-biotin-avidin-colloidal gold composite can be captured by corresponding stationary probe, and presents the red Radioactive colloidal gold detection signal of male in corresponding band position.
7. according to the described silver-colored recovering signal reinforcement of claim 1, it is characterized in that carrying out earlier the colloidal gold chromatographic colour developing, redye by silver-colored developing solution (silver lactate developing solution, Silver Nitrate developing solution) again, make its silver be reduced the formation black particle, make signal strengthen 10～100 times at the Radioactive colloidal gold position.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2006069537A1 (en) * | 2004-12-29 | 2006-07-06 | Suzhou Yangze Delta Academy Of Bio-X Science Ltd. | The optimum method of amplification on polymerase chain reaction |
| CN103242547A (en) * | 2013-01-20 | 2013-08-14 | 温州医学院 | Biomacromolecule cross-linking agent for membranes as well as preparation method and applications thereof |
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2001
- 2001-04-13 CN CN 01105979 patent/CN1381589A/en active Pending
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2006069537A1 (en) * | 2004-12-29 | 2006-07-06 | Suzhou Yangze Delta Academy Of Bio-X Science Ltd. | The optimum method of amplification on polymerase chain reaction |
| CN103242547A (en) * | 2013-01-20 | 2013-08-14 | 温州医学院 | Biomacromolecule cross-linking agent for membranes as well as preparation method and applications thereof |
| CN103242547B (en) * | 2013-01-20 | 2018-02-02 | 温州医科大学 | A kind of film boiomacromolecule crosslinking agent and preparation method and application |
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