CN103952493A - Application of fluorescent in-situ hybridization polyclonal separating probe for renal carcinoma and kit thereof - Google Patents
Application of fluorescent in-situ hybridization polyclonal separating probe for renal carcinoma and kit thereof Download PDFInfo
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Abstract
The invention relates to application of a fluorescent in-situ hybridization polyclonal separating probe for renal carcinoma and a kit thereof. Clonal fragments used by the polyclonal separating probe provided by the invention are respectively a combination of RP11-416B14, CTD-2522M13 and CTD-2516D6, and a combination of CTD-2312C1, CTD-2248C21 and RP11-959H17. According to the invention, the defect that karyotype analysis can not be performed conventionally after tumor cells are subjected to cell culture until a mitotic phase in the prior art is overcome. The typical gene modification in Xp11.2 translocation/TFE3 gene fusion related renal carcinoma is detected by using the FISH technology, thereby diagnosing the tumor. The fluorescent in-situ hybridization polyclonal separating probe application is high in accuracy, high in specificity, high in success rate, strong in fluorescent signals and convenient to operate. The invention can be applied in paraffin sections, widens the scope of detection specimens, and greatly optimizes the diagnosis method of Xp11.2 translocation/TFE3 gene fusion related renal carcinoma.
Description
Technical field
The invention belongs to the Application Areas of fluorescence in situ hybridization probe, particularly the fluorescence in situ hybridization polyclone separate probe of kidney and test kit application thereof.
Background technology
So-called kidney refers to Xp11.2 transposition/TFE3 gene fusion dependency kidney, it is the different subtypes renal cell carcinomas of WHO rebaptism in 2004, there is fracture transposition with TFE3 gene on XP11.2 site, and be fused to feature with the genes involved such as APSL, PRCC, PSF, NonO, CLTC, cause the unconventionality expression taking TFE3 albumen as main Regulated Proteins.Xp11.2 transposition/TFE3 gene fusion dependency kidney is mainly apt to occur in teenager, accounts for 1/3 of renal cell carcinoma in children, and adult patients is rare.Because its sickness rate is lower, still insufficient for the understanding of Xp11.2 transposition/TFE3 gene fusion dependency kidney at present, prognosis is poor, and adult patients is poorer compared with children's prognosis, mainly with pathological, tumor grade by stages, whether have distant metastasis etc. relevant.Xp11.2 transposition/TFE3 gene fusion dependency is invalid to conventional immunotherapy, also insensitive to chemicotherapy, and operation is its main therapeutic modality, and patient is prone to lymphoglandula and distant metastasis, only has targeted therapy to be proved to be certain effect.So Accurate Diagnosis is most important to judging patient's prognosis judgement and postoperative assisting therapy.
The distinctive change of tool of Xp11.2 transposition/TFE3 gene fusion dependency kidney is TFE3 gene generation balanced translocation on Xp11.2 site.At present known have 8 kinds of multi-form transpositions and a fusion, 5 kinds can clearly know gene fusion site respectively: t (X; 1) (p11.2; Q21) PRCC causing and TFE3 gene fusion, t (X; 17) (p11.2; Q25) ASPL causing and TFE3 gene fusion, t (X; 1) (p11.2; P34) PSF causing and TFE3 gene fusion, t (X; 17) (p11.2; Q23) CLTC causing and TFE3 gene fusion and inv (X) (p11; Q12) NonO (p54 causing
nrb).But gene fusion in any shape, all causes the change of TFE3 promotor, thereby cause the unconventionality expression taking TFE3 albumen as main modulin.Therefore, by the method for immunohistochemical methods, be, the main method of current diagnosis Xp11.2 transposition/TFE3 gene fusion dependency kidney to the detection of TFE3 albumen.But ImmunohistochemistryMethods Methods is vulnerable to more multifactorial impact, may be because draw materials improper or use BouinShi liquid to cause false negative to occur; Also may be because false positive results appears in the reasons such as excessively antigen recovery, too high, the excessive signal amplification of antibody concentration.TFE3 albumen is not only specific expressed in Xp11.2 transposition/TFE3 gene fusion dependency kidney in addition, also there will be the positive findings of TFE3 immunohistochemical methods in the tumor tissues such as granular cell tumor, adrenocortical carcinoma, Perivascular epithelioid cell tumor and high-level myxofibrosarcoma.The method that karyotyping and RT-PCR detect is to make a definite diagnosis the gold standard of XP11.2 transposition/TFE3 gene fusion dependency kidney.But in clinical position, cannot conventional carry out again karyotyping to the capable cell cultures of tumour cell to mitotic division after date, and RT-PCR extracts RNA and is subject to several factors restriction and technical sophistication to be not suitable for widespread use from paraffin section, thus need at present one badly can be fast, the method for economy, Accurate Diagnosis Xp11.2 transposition/TFE3 gene fusion dependency kidney.
Summary of the invention
The object of the invention is the defect for above-mentioned diagnostic method, a kind of fluorescence in situ hybridization polyclone separate probe and test kit application thereof of kidney are provided.
Technical scheme of the present invention is:
The fluorescence in situ hybridization polyclone separate probe of kidney, its technical characteristics is that the cloned sequence that polyclone separate probe is selected is respectively RP11-416B14, CTD-2522M13 and CTD-2516D6 combination, and CTD-2312C1, CTD-2248C21 and RP11-959H17 combination.
Described polyclone separate probe, wherein RP11-416B14, CTD-2522M13 and CTD-2516D6 integrated positioning are in TFE3 telomere side, and three is labeled as identical green fluorescence signal.
Described polyclone separate probe, wherein CTD-2312C1, CTD-2248C21 and RP11-959H17 integrated positioning are in TFE3 kinetochore side, and three is labeled as identical red fluorescence signal.
The color of the cloned sequence combination institute mark of the described TFE3 of being positioned at telomere side, TFE3 kinetochore side is respectively green and red.
Another technical scheme of the present invention is:
The fluorescence in situ hybridization polyclone separate probe test kit of kidney, this test kit by probe hybridization liquid and 4 ', 6-diamidino-2-phenylindone counterstain composition, its technical characteristics is:
(1) TFE3 telomere side BAC cloned sequence is the combination of RP11-416B14, CTD-2522M13 and CTD-2516D6, is labeled as green fluorescence; TFE3 kinetochore side BAC cloned sequence is the combination of CTD-2312C1, CTD-2248C21 and RP11-959H17, is labeled as red fluorescence;
(2) probe hybridization liquid is to be mixed in proportion configuration by described polyclone separate probe and Human Cot-1DNA, hybridization buffer, purified water to form, and needs-20 DEG C of freezing preservations of lucifuge;
(3) 4 ', 6-diamidino-2-phenylindone counterstain is mainly used in nucleus DNA dyeing.
Described TFE3 telomere side BAC cloned sequence is the combination of RP11-416B14, CTD-2522M13 and CTD-2516D6, is labeled as green fluorescence; TFE3 kinetochore side BAC cloned sequence is the combination of CTD-2312C1, CTD-2248C21 and RP11-959H17, be labeled as red fluorescence, and above-mentioned polyclone separate probe and Human Cot-1DNA, hybridization buffer, purified water are mixed in proportion and are configured to probe hybridization liquid.
The present invention also has a kind of technical scheme to be:
The fluorescence in situ hybridization polyclone separate probe test kit application of kidney, its technical characteristics is the application in diagnosis Xp11.2 transposition/TFE3 gene fusion dependency kidney.
Thereby advantage of the present invention and effect are to utilize FISH technology for detection Xp11.2 transposition/TFE3 gene fusion dependency kidney, distinctive gene alteration is diagnosed this tumour, the accuracy rate of this fluorescence in situ hybridization polyclone separate probe application is high, specificity is high, success ratio is high, fluorescent signal is strong, simple to operation, can be applicable in paraffin section, expand the scope that detects sample, greatly optimized the diagnostic method of Xp11.2 transposition/TFE3 gene fusion dependency kidney.
Brief description of the drawings
Fig. 1---fluorescence in situ hybridization polyclone separate probe station-keeping mode schematic diagram.
Fig. 2---women's Papillary Renal Cell Carcinoma negative control case application schematic diagram.
Fig. 3---male sex's Papillary Renal Cell Carcinoma negative control case application schematic diagram.
Fig. 4---women's clear cell carcinoma of kidney negative control case application schematic diagram.
Fig. 5---the transparent renal cell carcinoma negative control of male sex's kidney case application schematic diagram.
Fig. 6---male sex Xp11.2 transposition/TFE3 gene fusion dependency kidney application schematic diagram.
Fig. 7---male sex Xp11.2 transposition/TFE3 gene fusion dependency kidney application schematic diagram.
Fig. 8---women Xp11.2 transposition/TFE3 gene fusion dependency kidney application schematic diagram.
Fig. 9---women Xp11.2 transposition/TFE3 gene fusion dependency kidney application schematic diagram.
Figure 10---women Xp11.2 transposition/TFE3 gene fusion dependency kidney application schematic diagram.
Figure 11---women Xp11.2 transposition/TFE3 gene fusion dependency kidney application schematic diagram.
Figure 12---women's case negates Xp11.2 transposition/TFE3 gene fusion dependency kidney pathological diagnosis schematic diagram.
Embodiment
Technical thought of the present invention is:
Fluorescence in situ hybridization be utilize be marked with fluorescein probe specificity combine with karyomit(e) and (or) gene locus, by the type of fluorescence microscope fluorescent signal, thereby detect the method that karyomit(e) and corresponding gene change, have that safe, quick, economic, highly sensitive, detection signal is strong, hybridization specificity is high, can show the advantages such as multiple color simultaneously, and made up traditional method to interval cell, complex karyotype cell and the defect that cannot diagnose of microdeletion.Meanwhile, fluorescence in situ hybridization technique can be applied on paraffin-embedded sample carries out retrospective study, greatly reduces the requirement to research sample.Based on the fast development of fluorescence in situ hybridization technique in recent years, the present invention, just according to the principle of fluorescence in situ hybridization, has proposed a kind of for diagnosing fluorescence in situ hybridization polyclone separate probe and the test kit application thereof of Xp11.2 transposition/TFE3 gene fusion dependency kidney.
Below by embodiment, the invention will be further elaborated.
The polyclone separate probe that the present invention adopts is different from prior art and existing probe, be based on to TFE3 gene two ends specific BAC cloned sequence binding site comprehensive analysis after the prioritization scheme that filters out.
In the present invention, TFE3 telomere side BAC cloned sequence is the combination of RP11-416B14 (fragment length is about 182Kb), CTD-2522M13 (fragment length is about 182Kb) and CTD-2516D6 (fragment length is about 218Kb); TFE3 kinetochore side BAC cloned sequence is the combination of CTD-2312C1 (fragment length is about 210Kb), CTD-2248C21 (fragment length is about 88Kb) and RP11-959H17 (fragment length is about 147Kb).These fragments are bacterial artificial chromosome clone (BAC clone), and its location on human chromosomal is all open.
Between TFE3 gene and selected, to be positioned at its both sides (telomere side and kinetochore side) BAC cloned sequence, maintain a certain distance and can be not overlapping, and the shortest in about 36Kb only between the BAC cloned sequence of two ends, two kinds of signals can be closely close in the time that transposition does not occur gene to make probe, can be because of design reasons, make two sitgnal distancels excessive and occur that signal separates.Respectively there is the BAC cloned sequence combination of 3 TFE3 gene both sides, require to exist a small amount of fragment overlapping between the adjacent cloned sequence of homonymy, prevent that homonymy cloned sequence from, because spacing is compared with occurring greatly signal fracture, causing the appearance of independent signal, cause the erroneous judgement of detected result.
In X chromosome TFE3 gene both sides, the BAC cloned sequence of 3 same colors of mark is in order to strengthen fluorescence intensity and scope respectively, improves the accuracy detecting.Each BAC cloned sequence selected in probe combinations is through test separately, the cloned sequence that finishing screen select-out signal intensity is strong, specificity is good.Although it is to be noted the cloned sequence length of the CTD-2248C21 88Kb that only has an appointment, but strength of signal is stronger in test, CTD-2248C21 both sides are connecting respectively CTD-2312C1 and RP11-959H17 simultaneously, disappearance CTD-2248C21 may cause the fracture of kinetochore side signal, therefore it is just selected as one of labeled fragment.
Select two groups totally 6 cloned sequences have following some advantage: (1) most cloned sequence sizes are substantially all in optimum mark lengths 150-200Kb left and right, not only strength of signal is similar each other for such cloned sequence, and there will not be because the cloned sequence strength difference of signal that causes different in size is larger; (2) between homonymy adjacent segment, exist a small amount of sequence overlapping, make color signal of the same race mutually continuous, there will not be the fracture of homonymy signal and the appearance of signal separately, reduce the probability of erroneous judgement; Between (3) two groups of cloned sequences, farthest at a distance of about 1100Kb, be less than 1500Kb, make two kinds of signals of TFE3 gene transposition does not occur time institute's mark occur merging, like this in the time occurring that signal separates for positive group can with feminine gender group formation sharp contrast.If spacing is far away,, all may there is separation signal in no matter whether transposition of gene, cannot realize the effect of probe; Shortest spacing between (4) two groups of cloned sequences is less, about 36Kb, and what on non-translocation chromosome, occur is to merge signal, is difficult for obscuring mutually with separation signal.
BAC cloned sequence mark fluorescent, fluorescence microscope easy to use, convenient directly perceived, meanwhile, if desired adopt other detection techniques also can be marked as the certification mark thing corresponding with it.
Effect of the present invention:
The present invention is according to the feature of the gene alteration of Xp11.2 transposition/TFE3 gene fusion dependency kidney, utilize fluorescence in situ hybridization principle, use respectively not homochromy fluorescently-labeled polyclone probe in TFE3 gene both sides, by detecting fusion or the separation signal in paraffin-embedded tissue section, judge whether tumor tissues gene specific group translocation occurs, improve accuracy rate of diagnosis, for judging that patient's prognosis and postoperative adjuvant therapy provide information and guidance accurately.According to our experimental result, the accuracy rate of this probe application is high, specificity is high, success ratio is high, experimental signal is strong, simple and quick, especially can in paraffin section, apply, expand the scope that detects sample, for Accurate Diagnosis Xp11.2 transposition/TFE3 gene fusion dependency kidney provides new instrument.
Embodiment 1:
Step 1: the preparation of polyclone DNA probe:
Search bacterial artificial chromosome (BAC clone) corresponding to X chromosome TFE3 gene both sides (being telomere side and kinetochore side) by http://genome.ucsc.edu/, 3 BAC cloned sequences that size is close are selected respectively in both sides, control maximum distance between both sides BAC cloned sequences in 1500Kb and non-overlapping copies, and 3 fragments of homonymy there is the overlapping of a fixed sequence each other.Choosing TFE3 telomere side BAC cloned sequence by above-mentioned requirements is CTD-2516D6 (chrX:48265836-48484403, fragment length is about 218Kb), CTD-2522M13 (chrX:48420425-48602847, fragment length is about 182Kb), RP11-416B14 (chrX:48580872-48763198, fragment length is about 182Kb); TFE3 kinetochore side BAC cloned sequence is CTD-2312C1 (chrX:48959513-49170367, fragment length is about 210Kb), CTD-2248C21 (chrX:49154756-49242991, fragment length is about 88Kb), RP11-959H17 (chrX:49293025-49440119, fragment length is about 147Kb).And at http://projects.tcag.ca/cgi-bin/efi sh/i ndex.cgi, selected BAC clone is carried out to specificity analyses, specify the specificity of selected BAC fragment on X chromosome.Fluorescence in situ hybridization polyclone separate probe station-keeping mode in the present invention as shown in Figure 1.Purchase corresponding BAC clone from Invitrogen company, after being cultivated, BAC clone extracts plasmid, utilize nick-translation, 3 BAC cloned sequences of telomere side are labeled as to green fluorescence with Sptectrum green-dUTP, side 3 BAC cloned sequences in kinetochore are labeled as to red fluorescence with Sptectrum orange-dUTP, and two ends color can be exchanged.And probe good mark and Human Cot-1DNA, hybridization buffer, purified water are configured to probe hybridization liquid in proportion ,-20 DEG C of freezing preservations of lucifuge.
Next step, adopt the method for fluorescence in situ hybridization by the probe of preparation, in the Xp11.2 of clinical diagnosis transposition/TFE3 gene fusion dependency kidney and non-Xp11.2 group translocation renal carcinoma tissue, verifies its diagnosis effect.
Step 2: fluorescence in situ hybridization process:
3um tumor tissue section is placed in to the roasting sheet of 65 ± 1 DEG C of thermostat containers spends the night.After taking-up, put into dimethylbenzene and soak 30min, then put into soaked in absolute ethyl alcohol 10min.Then put into 100%, 85%, 70% gradient ethanol and the each 3min of purified water rehydration, then put into 100 ± 5 DEG C of purified water and boil sheet 20min.After taking-up is dried, drip 100ul stomach en-reaction solution in sample areas, digestion 5min.Put into rapidly 2 × SSC washing 5min, finally put into room temperature 70%, 85%, 100% gradient ethanol each 3min that dewaters successively, taking-up is dried.Add the hybridization solution of 10 μ l to dry 22 × 22mm cover glass, by sample chips upset, make sample down, cover rapidly, after reversion, light cap slide is uniformly distributed hybridization solution; Along cover glass edge mounting, cover the edge that cover glass contacts with slide glass with rubber cement completely; Slide is placed on the hot platform of 85 ± 1 DEG C to sex change 5min; Rapidly slide is put into the hybridizing box of preheating, lucifuge, 37 ± 1 DEG C of overnight incubation.2xSSC solution, 0.1%NP-40/2xSSC solution are put into the water-bath preheating of 37 ± 1 DEG C; Remove rubber cement and cover glass, slide is put into 2xSSC solution and wash 10min; Proceed to again and in 0.1%NP-40/2xSSC solution, wash 5mi n; Then room temperature 70% ethanol dehydration 3min.After taking-up slide in the dark dries, drip 10 μ l DAPI counterstains to dry 22 × 22mm cover glass, reversion sample chips, contacts the target area of cover glass slide glass, and after reversion, light pressure, avoids producing bubble, in the dark deposits, to be seen.
Result is judged:
Use standard with reference to current double-colored probe, in darkroom, excite with Olympus B × 5I fluorescent microscope DAPI/FIFC/TexasRed tri-look optical filterings, 100 times of thing Microscopic observation interval cells, differentiate fluorescent signal; The immunofluorescence software analysis image providing with Imstar company, assesses the probe hybridization situation of whole section.The whole nucleus of each example section has the nucleus of 100 non-overlapping copies at least, and observes clear and definite fluorescent signal, could determine that immunofluorescence in situ hybridization detects effectively.
Use fluorescent microscope can observe two kinds of signal types: (1) merges signal: not occurring in the tumor tissues of TFE3 group translocation, because double-colored signal is at a distance of the yellow signal compared with closely occurring (effect of red and green color stack) or red green continuous signal.The quantity that merges signal depends on X chromosome number in nucleus, and normal female occurs that 2 merge signal, and the male sex only occurs that 1 merges signal; (2) separation signal: in the time there is the transposition of TFE3 genic balance, TFE3 gene telomere lateral translocation is to other karyomit(e), and after transposition X chromosome only retain former TFE3 kinetochore side group because of, separating appears in the red and green signals that causes being marked at respectively TFE3 gene two ends.Only have in the time that red and green signals separation occurs simultaneously and be above apart from a signal diameter, just can be designated as separation signal.Women Xp11.2 transposition/TFE3 gene fusion dependency patients with renal cell carcinoma can occur that 1 merges signal and 1 pair of separation signal, and male patient only there will be 1 pair of separation signal.
The whole nucleus of each example section has the nucleus of 100 non-overlapping copies at least, has the red green separation signal of neoplastic cell nuclei appearance that exceedes 10% at nucleus, judges the fluorescence in situ hybridization positive; There is not red green separation signal, judge fluorescence in situ hybridization feminine gender; Occur that separately redness or green do not count.
Result:
We utilize this polyclone separate probe to detect to 30 routine tumor of kidney samples, and wherein 10 routine clinical diagnosises are Xp11.2 transposition/TFE3 gene fusion dependency kidney, 10 routine corpora mammillaria renal cell carcinomas, 10 routine clear cell carcinoma of kidney.If there is separation signal and reach fluorescence in situ hybridization diagnosis positive criteria, can make a definite diagnosis Xp11.2 transposition/TFE3 gene fusion dependency kidney in patient's pathological section.In 10 routine Xp11.2 transposition/TFE3 gene fusion dependency kidneys, 9 examples meet fluorescence in situ hybridization Case definition, are judged as the positive, and 1 example is negative; Corpora mammillaria renal cell carcinoma and renal clear cell carcinoma section all do not detect separation signal, are judged as feminine gender.Fig. 2 is women's Papillary Renal Cell Carcinoma negative control case, sees 2 red green fusion signals in tumor tissue cell's core, represents 2 X chromosomes that transposition does not occur; Fig. 3 is male sex's Papillary Renal Cell Carcinoma negative control case, sees 1 red green fusion signal in tumor tissue cell's core, represents 1 X chromosome that transposition does not occur; Fig. 4 is women's clear cell carcinoma of kidney negative control case, sees 2 red green fusion signals in tumor tissue cell's core, represents 2 X chromosomes that transposition does not occur; Fig. 5 is the transparent renal cell carcinoma negative control of male sex's kidney case, sees 1 red green fusion signal in tumor tissue cell's core, represents 1 X chromosome that transposition does not occur; Fig. 6 is male sex Xp11.2 transposition/TFE3 gene fusion dependency kidney, sees 1 pair of red green separation signal in tumor tissue cell's core, and the X chromosome of transposition occurs in representative; Fig. 7 is male sex Xp11.2 transposition/TFE3 gene fusion dependency kidney, sees 1 pair of red green separation signal in tumor tissue cell's core, and the X chromosome of transposition occurs in representative; Fig. 8 is women Xp11.2 transposition/TFE3 gene fusion dependency kidney, sees that 1 pair of red green separation signal and 1 merges signal in tumor tissue cell's core, represents that respectively the X chromosome of transposition does not occur for 1 X chromosome that transposition occurs and 1; Fig. 9 is women Xp11.2 transposition/TFE3 gene fusion dependency kidney, sees that 1 pair of red green separation signal and 1 merges signal in tumor tissue cell's core, represents that respectively the X chromosome of transposition does not occur for 1 X chromosome that transposition occurs and 1; Figure 10 is women Xp11.2 transposition/TFE3 gene fusion dependency kidney, sees that 1 pair of red green separation signal and 1 merges signal in tumor tissue cell's core, represents that respectively the X chromosome of transposition does not occur for 1 X chromosome that transposition occurs and 1; Figure 11 is women Xp11.2 transposition/TFE3 gene fusion dependency kidney, sees that 1 pair of red green separation signal and one merges signal in tumor tissue cell's core, represents that respectively the X chromosome of transposition does not occur for 1 X chromosome that transposition occurs and 1; Figure 12 is women's case, clinical pathology and immunodiagnosis are Xp11.2 transposition/TFE3 gene fusion dependency Diagnosis of Renal Cell Carcinoma schematic diagram, but this fluorescence in situ hybridization polyclone separate probe detects negative, in tumor tissue cell's core, see 2 pairs of red green fusion signals, representing 2 X chromosomes that transposition does not occur, negates pathology and immunodiagnosis thus.
Evaluate:
Two groups of 6 cloned sequences that this probe adopts not yet have same probe to use, and cloned sequence size is relatively consistent.Each cloned sequence marking signal intensity is more similar, being interrupted does not appear in the probe signals of homonymy mark, the more general probe of strength of signal is strong, specificity is high, the reliability and the validity that fully take into account the probe when being applied to diagnostic reagent and being prepared into diagnostic kit, meet the requirement that is applied to clinical diagnosis.Utilize fluorescence in situ hybridization technique, use TFE3 gene double-colored separation fluorescence in situ hybridization polyclone probe diagnostics Xp11.2 transposition/TFE3 gene fusion dependency kidney accurately, fast, economy and success ratio high, optimized greatly the diagnostic method of Xp11.2 transposition/TFE3 gene fusion dependency kidney.From accompanying drawing 2-5, can find out that it is all negative utilizing all corpora mammillaria renal cell carcinomas of this probe in detecting and clear cell carcinoma of kidney, and in accompanying drawing 6-11, show in the tumor tissue section of this probe in detecting Xp11.2 transposition/TFE3 gene fusion dependency kidney and all can show fluorescent signal in most nucleus, and fluorescent signal is clear, luminance signals is high, and all successfully carries out FISH detection in each routine tissue slice.Experimental result shows that the present invention is in high specificity and the high advantage of susceptibility, and this probe penetrance is strong simultaneously, and signal is clear, and luminance signals is high meets the requirement that is prepared into diagnostic kit.
Embodiment 2:Xp11.2 transposition/TFE3 gene fusion dependency Diagnosis of Renal Cell Carcinoma test kit
The fluorescence in situ hybridization polyclone separate probe test kit of kidney, this test kit by probe hybridization liquid and 4 ', 6-diamidino-2-phenylindone counterstain composition, is characterized in that:
(1) TFE3 telomere side BAC cloned sequence is the combination of RP11-416B14, CTD-2522M13 and CTD-2516D6, is labeled as green fluorescence; TFE3 kinetochore side BAC cloned sequence is the combination of CTD-2312C1, CTD-2248C21 and RP11-959H17, is labeled as red fluorescence.
(2) by two kinds of polyclone separate probe be mixed in proportion and be configured to probe hybridization liquid with Human Cot-1DNA, hybridization buffer, purified water ,-20 DEG C of freezing preservations of lucifuge.
(3) 4 ', 6-diamidino-2-phenylindone counterstain is mainly used in nucleus DNA dyeing.
The fluorescence in situ hybridization polyclone separate probe test kit of this kidney is mainly used in diagnosing Xp11.2 transposition/TFE3 gene fusion dependency kidney, it is characterized in that: TFE3 telomere side BAC cloned sequence is the combination of RP11-416B14, CTD-2522M13 and CTD-2516D6, is labeled as green fluorescence; TFE3 kinetochore side BAC cloned sequence is the combination of CTD-2312C1, CTD-2248C21 and RP11-959H17, be labeled as red fluorescence, and above-mentioned polyclone separate probe and Human Cot-1DNA, hybridization buffer, purified water are mixed in proportion and are configured to probe hybridization liquid.
Detection kit using method of the present invention is as follows:
1. the sample that comprises tumor tissues is fixed through 10% neutral formalin, after paraffin embedding, be prepared into 3um tissue slice;
2. section is placed in to the roasting sheet of 65 ± 1 DEG C of thermostat containers and spends the night, after taking-up, put into dimethylbenzene and soak 30min, then put into soaked in absolute ethyl alcohol 10min.Then put into 100%, 85%, 70% gradient ethanol and the each 3min of purified water rehydration, then put into 100 ± 5 DEG C of purified water and boil sheet 20min;
3. after taking out and drying, drip 100ul stomach en-reaction solution in sample areas, digestion 5min, puts into rapidly 2 × SSC washing 5min, finally puts into room temperature 70%, 85%, 100% gradient ethanol each 3min that dewaters successively, and taking-up is dried;
4. add 10 μ l probe hybridization liquid of the present invention to dry 22 × 22mm cover glass, by sample chips upset, make sample down, cover rapidly, after reversion, light cap slide is uniformly distributed hybridization solution, along cover glass edge mounting, covers the edge that cover glass contacts with slide glass with rubber cement completely;
5. slide is placed on the hot platform of 85 ± 1 DEG C, sex change 5min, puts into the hybridizing box of preheating by slide rapidly, lucifuge, 37 ± 1 DEG C of overnight incubation;
6. preheating is carried out in the water-bath of 2xSSC solution, 0.1%NP-40/2xSSC solution being put into 37 ± 1 DEG C, remove rubber cement and cover glass in section, slide is put into 2xSSC solution and wash 10min, then proceed in 0.1%NP-40/2xSSC solution and wash 5min; Then room temperature 70% ethanol dehydration 3min;
7., after taking-up slide in the dark dries, drip 10 μ l DAPI counterstains to dry 22 × 22mm cover glass, reversion sample chips, make the target area contact of cover glass slide glass, after reversion, light pressure, avoids producing bubble, in the dark deposit, use fluorescence microscope fluorescent signal.In non-Xp11.2 transposition/TFE3 gene fusion dependency patients with renal cell carcinoma, red and green signals merges demonstration yellow signal, i.e. negative findings; In the tissue slice of Xp11.2 transposition/TFE3 gene fusion dependency patients with renal cell carcinoma, because fracture transposition occurs X chromosome TFE3 gene, red and green signals is separated, be judged as positive findings, thereby clarify a diagnosis.
Claims (7)
1. the fluorescence in situ hybridization polyclone separate probe of kidney, it is characterized in that the cloned sequence that polyclone separate probe is selected is respectively RP11-416B14, CTD-2522M13 and CTD-2516D6 combination, and CTD-2312C1, CTD-2248C21 and RP11-959H17 combination.
2. the fluorescence in situ hybridization polyclone separate probe of kidney according to claim 1, is characterized in that wherein RP11-416B14, CTD-2522M13 and CTD-2516D6 integrated positioning are in TFE3 telomere side, and three is labeled as identical green fluorescence signal.
3. the fluorescence in situ hybridization polyclone separate probe of kidney according to claim 1, is characterized in that wherein CTD-2312C1, CTD-2248C21 and RP11-959H17 integrated positioning are in TFE3 kinetochore side, and three is labeled as identical red fluorescence signal.
4. according to the fluorescence in situ hybridization polyclone separate probe of the kidney described in claim 2,3, it is characterized in that the cloned sequence combination institute marker color that is positioned at TFE3 telomere side, TFE3 kinetochore side is labeled as respectively green and redness.
5. the fluorescence in situ hybridization polyclone separate probe test kit of kidney, this test kit by probe hybridization liquid and 4 ', 6-diamidino-2-phenylindone counterstain composition, is characterized in that:
(1) TFE3 telomere side BAC cloned sequence is the combination of RP11-416B14, CTD-2522M13 and CTD-2516D6, is labeled as green fluorescence; TFE3 kinetochore side BAC cloned sequence is the combination of CTD-2312C1, CTD-2248C21 and RP11-959H17, is labeled as red fluorescence;
(2) probe hybridization liquid is to be mixed in proportion configuration by described polyclone separate probe and Human Cot-1DNA, hybridization buffer, purified water to form, and needs-20 DEG C of freezing preservations of lucifuge;
(3) 4 ', 6-diamidino-2-phenylindone counterstain is mainly used in nucleus DNA dyeing.
6. a fluorescence in situ hybridization polyclone separate probe test kit for kidney as claimed in claim 5, is characterized in that: TFE3 telomere side BAC cloned sequence is the combination of RP11-416B14, CTD-2522M13 and CTD-2516D6, is labeled as green fluorescence; TFE3 kinetochore side BAC cloned sequence is the combination of CTD-2312C1, CTD-2248C21 and RP11-959H17, be labeled as red fluorescence, and above-mentioned polyclone separate probe and Human Cot-1DNA, hybridization buffer, purified water are mixed in proportion and are configured to probe hybridization liquid.
7. the fluorescence in situ hybridization polyclone separate probe test kit of a kidney as claimed in claim 5 application, is characterized in that the application in diagnosis Xp11.2 transposition/TFE3 gene fusion dependency kidney.
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