CN113265465A - Kit for detecting methylation degree of gene promoter region of mouse retrovirus integration site 1 homolog relevant to cervical cancer and application of kit - Google Patents
Kit for detecting methylation degree of gene promoter region of mouse retrovirus integration site 1 homolog relevant to cervical cancer and application of kit Download PDFInfo
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Abstract
The invention discloses a kit for detecting methylation degree of a homologous gene promoter region of a murine retrovirus integration site 1 related to cervical cancer, wherein each kit comprises three pairs of methylation specific amplification primers and three methylation specific sequencing primers of the homologous gene promoter region of the murine retrovirus integration site 1, wherein an upstream primer has three nucleotide sequences, a downstream primer has three nucleotide sequences, the methylation specific sequencing primer has three nucleotide sequences, and the methylation level of the homologous gene promoter region of the murine retrovirus integration site 1 is in positive correlation with the prevalence rate of the cervical cancer to detect the cervical cancerMRVI1The diagnosis kit based on the methylation level of the gene promoter region can conveniently and quickly realize the detection of cervical cancer on the molecular level, has high detection efficiency and strong pertinence, and simultaneously usesMRVI1Medicine with gene promoter region methylation as target spotThe compound is expected to become a new means for auxiliary diagnosis, detection and screening of cervical cancer.
Description
Technical Field
The invention relates to a detection kit for auxiliary diagnosis of cervical cancer and application thereof, in particular to three detection kits for auxiliary diagnosis of cervical cancer and application thereof, and specifically relates to a kit for detecting methylation degree of a promoter region of a homologous gene of a murine retrovirus integration site 1 related to cervical cancer and application thereof.
Background
Cervical cancer (cervical cancer) is a malignant tumor that occurs in the female cervix, and is a relatively common disease with high mortality. Under the existing medical conditions, cervical cancer is difficult to completely cure, but if early discovery and early treatment can be achieved, most of the cervical cancer has better prognosis. At present, cervical cancer screening mainly adopts three methods, namely cervical scraping examination, cervical TCT smear examination and HPV detection, wherein the HPV detection has higher relative accuracy, and if the 3 examinations are all abnormal results, cervical biopsy can be generally performed to make clear diagnosis. Currently, HPV infection is the leading cause of cervical cancer, and other associated risk factors include genetics, multiple sexual partners, premature initiation of sexual life, multiple pregnancy and multiple birth, and immunodeficiency diseases. In the case of cervical cancer, the preferred treatment mode is hysterectomy, and the operation is followed by radiotherapy and chemotherapy, which is helpful for prolonging the survival time of the patient.
With the development of science and technology, genetic examination and treatment of tumors become the hot topic of the medical community, people find that there are proto-oncogenes and tumor suppressor genes in human cells, when these two genes are mutated due to some conditions, cancer will be generated, tumors are one of the manifestation forms of cancer, gene therapy is to eliminate cancer cells at fixed points, and essentially, cancer is a genetic disease, and the occurrence, development and recurrence of which are related to the mutation, deletion and malformation of genes. Cervical cancer is a complex disease caused by the combined action of genetic factors and environmental factors, and the search for cervical cancer related genes and further the elucidation of the genetic mechanism of the onset of cervical cancer has become a hotspot of current research. Although more and more medical research institutions pay attention to and develop etiology research of cervical cancer, and research mostly focuses on the correlation between the methylation level of the promoter region of the related candidate gene and the cervical cancer, the pathogenesis of the cervical cancer is still not completely elucidated, and the improvement of the prevention and treatment level of the cervical cancer is undoubtedly prevented.
The Murine Retrovirus Integration Site 1 Homolog (Murine Retrovirus Integration Site 1 Homolog, MRVI1) (chr 11:10, 573, 091-containing 10,693,988) gene is similar to the mouse tumor suppressor gene (Mrvi1), which is often disrupted by the mouse AIDS-related virus (MRV). The protein encoded by MRVI1(chr 11:10, 573,091-10,693,988) is a substrate for cGMP-dependent kinase 1 and can act as a regulator of IP 3-induced calcium release. Mouse studies have shown that MRV integration at Mrvi1 induces myeloid leukemia by altering the expression of genes important for myeloid cell growth and/or differentiation, and thus the genes may function as myeloid leukemia cancer suppressor genes.
At present, no kit for detecting methylation degree of a gene promoter region of a mouse retrovirus integration site 1 homolog related to cervical cancer and application related research reports are disclosed at home and abroad.
Disclosure of Invention
The technical problem to be solved by the invention is to provide a kit for detecting the methylation degree of a promoter region of a homologous gene of a murine retrovirus integration site 1 related to cervical cancer and an application thereof aiming at the current situation of the prior art, wherein the methylation level of the promoter region of the homologous gene of the murine retrovirus integration site 1 is positively related to the prevalence rate of the cervical cancer, the detection efficiency is high, and the pertinence is strong.
The technical scheme adopted by the invention for solving the technical problems is as follows:
the kit for detecting the methylation degree of the promoter region of the homologous gene of the murine retrovirus integration site 1 related to the cervical cancer comprises three pairs of methylation specific amplification primers and three methylation specific sequencing primers of the promoter region of the homologous gene of the murine retrovirus integration site 1, wherein
A first group:
the methylation specific forward primer has the nucleotide sequence:
5’-ttagtatttaagaggaaaagggaaagttgt-3’
the methylation specific downstream primer has the nucleotide sequence:
5’-biotin-cccccatcataactaaaaatct-3’
the methylation specific sequencing primer has the nucleotide sequence:
5’-gggaaagttgttggg-3’；
second group:
the methylation specific forward primer has the nucleotide sequence:
5’-tggggagtttttattatttaaggttaatg-3’
the methylation specific downstream primer has the nucleotide sequence:
5’-biotin-taaatcccaacccctctcaaa-3’
the methylation specific sequencing primer has the nucleotide sequence:
5’-aggttaatgttatatttggttt-3’；
third group:
the methylation specific forward primer has the nucleotide sequence:
5’-aagtatgtgagtttggagaaga-3’
the methylation specific downstream primer has the nucleotide sequence:
5’-biotin-tctcccaaaccattctctctaac-3’
the methylation specific sequencing primer has the nucleotide sequence:
5’-ggtaggggttgttttta-3’。
the technical scheme adopted also comprises:
the three groups of detection kits can be used for detecting the methylation degree of the gene promoter region of the mouse retrovirus integration site 1 homolog related to the cervical cancer singly or in combination; can be used as auxiliary diagnosis, detection or screening medicine for cervical cancer.
The application of the kit for detecting the methylation degree of the gene promoter region of the integration site 1 homolog of the murine retrovirus related to the cervical cancer is characterized in that: the method comprises the following steps:
the method comprises the following steps: carrying out bisulfite conversion on the sample DNA by adopting an EZ DNA Methylation Kit-Gold (EZ DNA Methylation-GoldTM Kit; ZYMO RESEARCH);
step two: adding 20ng of the DNA sample transformed in the step one into Zymo TaqTM PreMix enzyme (Zymo TaqTM PreMix, ZYMO RESEARCH), adding the pair of methylation specific amplification primers of the gene promoter region of the neurotrophic receptor tyrosine kinase 3, and carrying out PCR amplification, wherein the amplification conditions are as follows: firstly, denaturation at 95 ℃ for 3 min; then maintaining the temperature at 94 ℃ for 30 s; then carrying out extension reaction at 56 ℃ for 30s and 72 ℃ for 1min, and carrying out extension reaction at 72 ℃ for 7min for 40 cycles;
step three: the prophase preparation of pyrosequencing is that 2 mu L of reaction binding beads, 38 mu L of binding buffer solution and 40 mu L of PCR products are added into a 96-hole PCR reaction plate and fully and uniformly mixed for 10min at room temperature; starting a vacuum pump to absorb the binding beads and the PCR product suspension, and sequentially immersing the binding beads and the PCR product suspension in 70% ethanol, 0.2M NaOH and washing buffer solution for 5s respectively; turning off the vacuum pump, placing the binding beads on the probe and the PCR product in 40 μ L annealing buffer solution (containing 1.5 μ L of sequencing primer), denaturing at 85 deg.C for 2min, cooling to room temperature, and allowing the primer and the template to anneal and hybridize; calculating the dosage according to Pyrosequencing software sequence design information, and sequentially adding a substrate mixture, an enzyme mixture and four dNTPs (QIAGEN) into a reagent cabin; placing the reagent cabin and the 96-hole reaction plate into a Pyrosequencing detector (PyroMark Q96 ID, QIAGEN) for reaction;
step four: pyrosequencing, namely calculating the dosage according to Pyrosequencing software sequence design information, and sequentially adding a substrate mixture, an enzyme mixture and four dNTPs (QIAGEN) into a reagent cabin; then, the reagent chamber and the 96-well reaction plate were placed in a Pyrosequencing apparatus (PyroMark Q96 ID, QIAGEN) to perform a reaction. Methylation analysis was performed using Pyro Q-CpG software from a pyrosequencer.
Compared with the prior art, the invention has the advantages that: the invention discloses a kit for detecting the methylation degree of a gene promoter region of a homologous gene of a murine retrovirus integration site 1 related to cervical cancer for the first time and application thereof, wherein the hypermethylation level of the gene promoter region of the homologous gene of the murine retrovirus integration site 1 leads toMRVI1Low expression of genesThereby affecting the occurrence and development of cervical cancer. The kit for detecting the methylation degree of the promoter region of the homologous gene of the murine retrovirus integration site 1 related to the cervical cancer comprises three pairs of methylation specific amplification primers and three methylation specific sequencing primers of the promoter region of the homologous gene of the murine retrovirus integration site 1, wherein an upstream primer has three nucleotide sequences, a downstream primer has three nucleotide sequences, the methylation specific sequencing primer has three nucleotide sequences, the methylation level of the promoter region of the homologous gene of the murine retrovirus integration site 1 is positively correlated with the prevalence rate of the cervical cancer to detect the cervical cancerMRVI1The diagnosis kit based on the methylation level of the gene promoter region can conveniently and quickly realize the detection of cervical cancer on the molecular level, has high detection efficiency and strong pertinence, and simultaneously usesMRVI1The medicament taking methylation of the gene promoter region as a target spot is expected to become a new means for auxiliary diagnosis, detection and screening of cervical cancer.
Drawings
FIG. 1 is a schematic diagram of the specific positions of 7 CpG sites in the promoter region of the detected sequence MRVI1 gene, namely Chr11:10,594,638-Chr11:10,715,535;
FIG. 2 is a graph showing the methylation signal intensity of corresponding CpG sites in percent shown in gray shading as a result of methylation level detection;
FIG. 3 is a diagram showing the correlation between 7 CpG sites in the promoter region of MRVI1 gene.
Detailed Description
The invention is described in further detail below with reference to the accompanying examples.
As shown in FIGS. 1 to 3, kits for detecting the methylation degree of the promoter region of the homologous gene of murine retrovirus integration site 1 associated with cervical cancer, each kit comprising three pairs of methylation specific amplification primers and three methylation specific sequencing primers for the promoter region of the homologous gene of murine retrovirus integration site 1, wherein
A first group:
the methylation specific forward primer has the nucleotide sequence:
5’-ttagtatttaagaggaaaagggaaagttgt-3’
the methylation specific downstream primer has the nucleotide sequence:
5’-biotin-cccccatcataactaaaaatct-3’
the methylation specific sequencing primer has the nucleotide sequence:
5’-gggaaagttgttggg-3’；
second group:
the methylation specific forward primer has the nucleotide sequence:
5’-tggggagtttttattatttaaggttaatg-3’
the methylation specific downstream primer has the nucleotide sequence:
5’-biotin-taaatcccaacccctctcaaa-3’
the methylation specific sequencing primer has the nucleotide sequence:
5’-aggttaatgttatatttggttt-3’；
third group:
the methylation specific forward primer has the nucleotide sequence:
5’-aagtatgtgagtttggagaaga-3’
the methylation specific downstream primer has the nucleotide sequence:
5’-biotin-tctcccaaaccattctctctaac-3’
the methylation specific sequencing primer has the nucleotide sequence:
5’-ggtaggggttgttttta-3’。
the technical scheme adopted also comprises:
the three groups of detection kits can be used for detecting the methylation degree of the gene promoter region of the mouse retrovirus integration site 1 homolog related to the cervical cancer singly or in combination; can be used as auxiliary diagnosis, detection or screening medicine for cervical cancer.
The technical scheme of the invention is supportively explained by aiming at specific embodiments.
1. Collection of study subjects
Cervical cancer patients were collected from a third hospital in Wenzhou (all confirmed by cervical biopsy). The screened patients have not received radiotherapy, chemotherapy or hormone treatment before diagnosis, do not combine other malignant tumors and serious internal diseases, and finally 9 cervical cancer patients are determined (48.90 +/-10.96 years old). Clinical tissues were collected with informed consent from all subjects and approved by the ethical committee of the second hospital affiliated with the university of medical science, wenzhou. Respectively collecting cervical cancer and matched tissues beside the cancer (more than or equal to 2cm from the edge of the tumor) in the operation process, shearing fresh tissue samples, washing the fresh tissue samples with physiological saline, putting the fresh tissue samples into a freezing tube, and quickly storing the frozen tissue samples in a liquid nitrogen tank for uniformly extracting genome DNA from the samples. 2. Extraction of genomic DNA
Extracting the genomic DNA of the sample obtained in the above step with a genomic DNA extraction kit (QIAGEN), and detecting the concentration of the obtained DNA with an Infinite F200 deacon microplate reader (Tecan, Switzerland) for useMRVI1And (3) detecting the DNA methylation level of the gene promoter region. 3. DNA methylation level determination
The present study adopted pyrosequencing technology pairMRVI1DNA methylation level analysis was performed at 7 CpG sites in the promoter region of the gene as shown in FIG. 1. The basic principle of this technique: after bisulfite treatment of the DNA sample, Polymerase Chain Reaction (PCR) amplification is used to maintain the methylated cytosine (C) base unchanged while the unmethylated C is converted to uracil (U), and PCR sequencing is performed using sequencing primers to determine which site is methylated. In the research, PyroMark Assay design2.0 software is adopted for primer design, and PCR amplification primers and sequencing primers used for experiments are as follows:
a first pair:
(1) methylation specific Forward primer (Forward primer)
5’-GGGGATTGTTATTTGTTGTGTGGATAT-3’(SEQ ID NO.1)，
(2) Methylation specific downstream primer (Reverse primer)
5’-Biotin-AAACTCCTCTAAAAACCCCACTC-3’(SEQ ID NO.2)，
(3) Methylation specific Sequencing primer (Sequencing primer)
5’-AGGGGTTTAGGGTGA-3’(SEQ ID NO.3)，
The second pair:
(4) methylation specific Forward primer (Forward primer)
5’-TGGGGAGTTTTTATTATTTAAGGTTAATG-3’(SEQ ID NO.4)，
(5) Methylation specific downstream primer (Reverse primer)
5’-Biotin-TAAATCCCAACCCCTCTCAAA-3’(SEQ ID NO.5)，
(6) Methylation specific Sequencing primer (Sequencing primer)
5’-AGGTTAATGTTATATTTGGTTT-3’(SEQ ID NO.6)，
And a third pair:
(7) methylation specific Forward primer (Forward primer)
5’-AAGTATGTGAGTTTGGAGAAGA-3’(SEQ ID NO.7)，
(8) Methylation specific downstream primer (Reverse primer)
5’-Biotin-TCTCCCAAACCATTCTCTCTAAC-3’(SEQ ID NO.8)，
(9) Methylation specific Sequencing primer (Sequencing primer)
5’-GGTAGGGGTTGTTTTTA-3’(SEQ ID NO.9)。
The specific experimental steps are as follows:
the method comprises the following steps: bisulfite conversion of sample DNA was performed using EZ DNA Methylation Kit-Gold (EZ DNA Methylation-GoldTM Kit; ZYMORESEARCH).
Step two: and B, taking 20ng of the DNA sample converted in the step A, adding the DNA sample into Zymo TaqTM PreMix enzyme (Zymo TaqTM PreMix, ZYMO RESEARCH), adding the pair of methylation specific amplification primers of the gene promoter region of the neurotrophic receptor tyrosine kinase 3, and carrying out PCR amplification under the amplification conditions: firstly, denaturation at 95 ℃ for 3 min; then maintaining the temperature at 94 ℃ for 30 s; then carrying out extension reaction at 56 ℃ for 30s and 72 ℃ for 1min, and carrying out extension reaction at 72 ℃ for 7min for 40 cycles;
step three: the prophase preparation of pyrosequencing is that 2 mu L of reaction binding beads, 38 mu L of binding buffer solution and 40 mu L of PCR products are added into a 96-hole PCR reaction plate and fully and uniformly mixed for 10min at room temperature; starting a vacuum pump to absorb the binding beads and the PCR product suspension, and sequentially immersing the binding beads and the PCR product suspension in 70% ethanol, 0.2M NaOH and washing buffer solution for 5s respectively; turning off the vacuum pump, placing the binding beads on the probe and the PCR product in 40 μ L annealing buffer solution containing 1.5 μ L of sequencing primer, denaturing at 85 deg.C for 2min, cooling to room temperature, and annealing and hybridizing the primer and the template; calculating the dosage according to Pyrosequencing software sequence design information, and sequentially adding a substrate mixture, an enzyme mixture and four dNTPs (QIAGEN) into a reagent cabin; the reagent chamber and the 96-well reaction plate were placed in a Pyrosequencing apparatus (PyroMark Q96 ID, QIAGEN) for reaction.
Step four: pyrosequencing, namely calculating the dosage according to Pyrosequencing software sequence design information, and sequentially adding a substrate mixture, an enzyme mixture and four dNTPs (QIAGEN) into a reagent cabin; then, the reagent chamber and the 96-well reaction plate were placed in a Pyrosequencing apparatus (PyroMark Q96 ID, QIAGEN) to perform a reaction. Methylation analysis was performed using Pyro Q-CpG software from a pyrosequencer (see FIG. 2 for an example of the results of methylation level detection).
4. Data analysis
In this study, the data is sorted and analyzed by using SPSS 23.0, and we find that: between cancerous and paracancerous tissue of the cervixMRVI1There was a difference in the DNA methylation level in the promoter region of the gene, the methylation level was higher in the cervical cancer tissue, and there was a difference in 7 CG sites (1)PValues of 0.001, 0.004, 0.005, 0.015, 0.001, 3.12E-04, 0.002), respectively). In addition, we found that there was a correlation between 7 sites (FIG. 3), therefore, we calculated the mean between the two groups and found that the methylation levels in cancer tissues were still higher than in paracancerous tissues after comparison of the mean between the two groups: (C.) (P=0.002)。
The kit for detecting the methylation degree of the promoter region of the homologous gene of the murine retrovirus integration site 1 related to the cervical cancer and the application thereof have the advantages of accuracy, reliability, flexibility, rapidness, economy and economy. The invention adopts the kit pairMRVI1The methylation level of the gene promoter region is detected, so that reference can be rapidly and reliably provided for auxiliary diagnosis, detection or screening of drugs for cervical cancer.
TABLE 1
| Grouping | Cancer (carcinoma) | Paracarcinoma | P Value of |
| MRVI1 | |||
| CG1 | 70.54 ± 22.47 | 33.37 ± 10.04 | 0.001 |
| CG2 | 50.96 ± 18.40 | 26.61 ± 6.77 | 0.004 |
| CG3 | 1.68 ± 0.17 | 1.45± 0.08 | 0.005a |
| CG4 | 89.70 (55.56, 96.70) | 45.20 (42.80, 54.30) | 0.015b |
| CG5 | 51.26 ± 16.02 | 23.69 ± 6.81 | 0.001 |
| CG6 | 50.81 ± 13.95 | 24.19 ± 7.27 | 3.12E-04 |
| CG7 | 51.21 ± 18.44 | 25.30 ± 7.47 | 0.002 |
| Mean MRVI1methylation (%) | 57.6 ± 17.62 | 30.16 ± 7.28 | 0.002 |
Note: is thickened byPThe value is less than 0.05, and the statistical significance is achieved; adding a means that the addition has statistical significance after logarithmic transformation (log 10); plus b indicates that non-parametric rank-sum test is applied.
Microliter metering remarks: 1L =1000ml 1ml = 1000. mu.L
The above description is not intended to limit the present invention, and the present invention is not limited to the above examples. Those skilled in the art will appreciate that various modifications, additions and substitutions are possible, without departing from the scope and spirit of the invention as disclosed in the accompanying claims.
Sequence listing
<110> second Hospital affiliated with Wenzhou medical university (Children's hospital affiliated with Wenzhou medical university)
<120> kit for detecting methylation degree of promoter region of homologous gene of murine retrovirus integration site 1 related to cervical cancer and application
<141> 2021-05-12
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tggggagttt ttattattta aggttaatg 29
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ggtaggggtt gttttta 17
Claims (3)
1. The kit for detecting the methylation degree of the promoter region of the homologous gene of the murine retrovirus integration site 1 related to the cervical cancer is characterized in that: each kit comprises three pairs of methylation specific amplification primers and three methylation specific sequencing primers of homologous gene promoter regions of the murine retrovirus integration site 1, wherein
A first group:
the methylation specific forward primer has the nucleotide sequence:
5’-ttagtatttaagaggaaaagggaaagttgt-3’
the methylation specific downstream primer has the nucleotide sequence:
5’-biotin-cccccatcataactaaaaatct-3’
the methylation specific sequencing primer has the nucleotide sequence:
5’-gggaaagttgttggg-3’；
second group:
the methylation specific forward primer has the nucleotide sequence:
5’-tggggagtttttattatttaaggttaatg-3’
the methylation specific downstream primer has the nucleotide sequence:
5’-biotin-taaatcccaacccctctcaaa-3’
the methylation specific sequencing primer has the nucleotide sequence:
5’-aggttaatgttatatttggttt-3’；
third group:
the methylation specific forward primer has the nucleotide sequence:
5’-aagtatgtgagtttggagaaga-3’
the methylation specific downstream primer has the nucleotide sequence:
5’-biotin-tctcccaaaccattctctctaac-3’
the methylation specific sequencing primer has the nucleotide sequence:
5’-ggtaggggttgttttta-3’。
2. the kit for detecting the methylation degree of the promoter region of the homologous gene of the murine retrovirus integration site 1 related to cervical cancer according to claim 1, wherein: the three groups of detection kits can be used for detecting the methylation degree of the gene promoter region of the mouse retrovirus integration site 1 homolog related to the cervical cancer singly or in combination; can be used as auxiliary diagnosis, detection or screening medicine for cervical cancer.
3. The application of the kit for detecting the methylation degree of the gene promoter region of the integration site 1 homolog of the murine retrovirus related to the cervical cancer is characterized in that: the method comprises the following steps:
the method comprises the following steps: carrying out bisulfite conversion on the sample DNA by adopting an EZ DNA Methylation Kit-Gold (EZ DNA Methylation-GoldTM Kit; ZYMO RESEARCH);
step two: adding 20ng of the DNA sample transformed in the step one into Zymo TaqTM PreMix enzyme (Zymo TaqTM PreMix, ZYMO RESEARCH), adding the pair of methylation specific amplification primers of the gene promoter region of the neurotrophic receptor tyrosine kinase 3, and carrying out PCR amplification, wherein the amplification conditions are as follows: firstly, denaturation at 95 ℃ for 3 min; then maintaining the temperature at 94 ℃ for 30 s; then carrying out extension reaction at 56 ℃ for 30s and 72 ℃ for 1min, and carrying out extension reaction at 72 ℃ for 7min for 40 cycles;
step three: the prophase preparation of pyrosequencing is that 2 mu L of reaction binding beads, 38 mu L of binding buffer solution and 40 mu L of PCR products are added into a 96-hole PCR reaction plate and fully and uniformly mixed for 10min at room temperature; starting a vacuum pump to absorb the binding beads and the PCR product suspension, and sequentially immersing the binding beads and the PCR product suspension in 70% ethanol, 0.2M NaOH and washing buffer solution for 5s respectively; turning off the vacuum pump, placing the binding beads on the probe and the PCR product in 40 μ L annealing buffer solution containing 1.5 μ L of sequencing primer, denaturing at 85 deg.C for 2min, cooling to room temperature, and annealing and hybridizing the primer and the template; calculating the dosage according to Pyrosequencing software sequence design information, and sequentially adding a substrate mixture, an enzyme mixture and four dNTPs (QIAGEN) into a reagent cabin; placing the reagent cabin and the 96-hole reaction plate into a Pyrosequencing detector (PyroMark Q96 ID, QIAGEN) for reaction;
step four: pyrosequencing, namely calculating the dosage according to Pyrosequencing software sequence design information, and sequentially adding a substrate mixture, an enzyme mixture and four dNTPs (QIAGEN) into a reagent cabin; then the reagent chamber and the 96-well reaction plate are placed into a Pyrosequencing detector (PyroMark Q96 ID, QIAGEN) for reaction, and methylation analysis is carried out by using Pyro Q-CpG software carried by a Pyrosequencing instrument.
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| US20070154931A1 (en) * | 2005-12-15 | 2007-07-05 | Radich Jerald P | Genes associate with progression and response in chronic myeloid leukemia and uses thereof |
| US20110318738A1 (en) * | 2008-12-23 | 2011-12-29 | University Of Utah Research Foundation | Identification and regulation of a novel dna demethylase system |
| EP2481813A1 (en) * | 2011-02-01 | 2012-08-01 | Centro di Riferimento Oncologico - Istituto Nazionale Tumori - Aviano | Markers of cutaneous melanoma and uses thereof |
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| US20070154931A1 (en) * | 2005-12-15 | 2007-07-05 | Radich Jerald P | Genes associate with progression and response in chronic myeloid leukemia and uses thereof |
| US20120072124A1 (en) * | 2005-12-15 | 2012-03-22 | Rosetta Inpharmatics, Llc | Genes associated with progression and response in chronic myeloid leukemia and uses thereof |
| US20110318738A1 (en) * | 2008-12-23 | 2011-12-29 | University Of Utah Research Foundation | Identification and regulation of a novel dna demethylase system |
| EP2481813A1 (en) * | 2011-02-01 | 2012-08-01 | Centro di Riferimento Oncologico - Istituto Nazionale Tumori - Aviano | Markers of cutaneous melanoma and uses thereof |
| US20130316931A1 (en) * | 2011-02-01 | 2013-11-28 | Centro Di Riferimento Oncologico-Instituto Nazionale Tumori-Aviano | Markers of melanoma and uses thereof |
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