CN107523615A - Brc3 hypotypes in PML RAR alpha fusion genes are identified using ddPCR methods - Google Patents
Brc3 hypotypes in PML RAR alpha fusion genes are identified using ddPCR methods Download PDFInfo
- Publication number
- CN107523615A CN107523615A CN201710505991.2A CN201710505991A CN107523615A CN 107523615 A CN107523615 A CN 107523615A CN 201710505991 A CN201710505991 A CN 201710505991A CN 107523615 A CN107523615 A CN 107523615A
- Authority
- CN
- China
- Prior art keywords
- added
- 13400rpm
- rna
- centrifugal column
- buffer
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6844—Nucleic acid amplification reactions
- C12Q1/6858—Allele-specific amplification
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6806—Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay
Landscapes
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Life Sciences & Earth Sciences (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Analytical Chemistry (AREA)
- Biophysics (AREA)
- Biochemistry (AREA)
- Microbiology (AREA)
- Molecular Biology (AREA)
- Biotechnology (AREA)
- Physics & Mathematics (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Immunology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
- Investigating Or Analysing Biological Materials (AREA)
Abstract
The invention discloses the Brc3 hypotypes in a kind of identification PML RAR alpha fusion genes using ddPCR methods, the ddPCR methods include the RNA in extraction blood and carry out QC assessments and RNA reverse transcriptions cDNA and carry out QC assessments.The present invention is directed to common Brc3 types, design detection fusion probes, and primer is expanded for fusion both sides sequence；Using RAR Α genes as internal reference, Ref probes are designed, primer only expands RAR Α genes.DdPCR detection method PML RAR α fusion bcr3 hypotypes in the present invention up to 0.01% abundance, the 0.1% of remote super RT PCR, reproducible, non-false positive；In the case of low AF, it is proposed that the increase of sample input amount, or repeat multiple operation repetitives.Due to the high sensitivity of the technology, therefore the early stage that can be applied to disease is made a definite diagnosis, dynamic monitoring and preventing and treating are recurred.
Description
Technical field
The invention belongs to gene identification method technical field, particularly relates to a kind of using the identification of ddPCR methods
Brc3 hypotypes in PML-RAR alpha fusion genes.
Background technology
PML-RAR alpha fusion genes are by t (15:17)(q22:Q21) transposition is formed, and is common in the white blood of acute progranulocyte
Sick (APL).RAR α and PML fusion have following characteristics：RAR α gene breaks site is located at intron 2；And PML bases
Because there is three regions to take part in t (15：17) transposition, i.e. introne 6 (Brc1, account for 55%), exon 6 (Brc3, accounts for
5%), 40%) introne 3 (brc3, accounts for.PML-RAR alpha fusion genes can be divided into three kinds of Asias according to the position of broken site
Type：As shown in Figure 1, elongated (L types, Brc1), anomaly (V-type, Brc3) and short (S types, brc3).Leukaemia is controlled at present
Treatment is more effective, and its patient is extended life cycle, but later stage follow-up finds that recurrence rate is higher in the several years, and possible cause is current
Using RT-PCR detection techniques, its detection sensitivity is only 0.1%.
The content of the invention
The present invention is in order to overcome the shortcomings of the prior art, there is provided one kind, which has, highly sensitive uses ddPCR methods
Identify the Brc3 hypotypes in PML-RAR alpha fusion genes.
The present invention is achieved by the following technical solutions：One kind is using ddPCR methods identification PML-RAR alpha fusion genes
In Brc3 hypotypes, the ddPCR methods include RNA in extraction blood and QC is assessed, and specific operation process is as follows：
(1) RNA is extracted, blood plasma is divided in 1.5ml EP pipes, often pipe dispenses 300 μ l；Using in Tiangeng kit from
The cell pyrolysis liquid CL of band, 750 μ l are added into blood, mixing of turning upside down, stand 1min；10000rpm centrifuges 1min；Abandon
Supernatant, stay precipitation；700 μ l CL are added into blood, is centrifuged under equal conditions, abandons supernatant, stay precipitation；
(2) 150 μ l buffer PKD are added, piping and druming mixes；Then 10 μ l Proteinase Ks are added, piping and druming mixes；Sample in
3min on ice is immediately placed in after 56 DEG C of thermostat, rotating speed 350rpm, 15min；13400rpm centrifuges 15min, transfer supernatant 160 μ
In l to 1.5ml EP pipes；Supernatant is placed in 80 DEG C of incubations 15min, 13400rpm on thermostat and quickly centrifuges 30s；Add 320 μ l
Buffer RLT, piping and druming mix, and add the ethanol of 1000 μ l 96~100%, are vortexed and mix；
(3) 700 μ l samples of transfer into RNeasy MinElute centrifugal columns, 13400rpm centrifugation 15s, abandon filtrate every time,
This step is repeated to finish until all samples are crossed into post；350 μ l buffer FRN are added to centrifuge 15s to centrifugal column, 13400rpm,
Abandon filtrate；
(4) mix is matched somebody with somebody:10 μ l DNaseI and 70 μ l buffer RDD, piping and druming mix, the film being added into centrifugal column
On, it is placed in 15min at a temperature of 20~30 DEG C of room temperature；500 μ l buffer FRN are added 13400rpm centrifugation 15s, to be protected to centrifugal column
Reserved filtrate；A new 2ml collecting pipe is changed, filtrate is rejoined into centrifugal column, 13400rpm centrifugation 15s, abandons filtrate；
(5) 500 μ l buffer RPE are added to centrifugal column, 13400rpm centrifugation 15s, filtrate is abandoned, is repeated once；Will be from
Stem is placed in new 2ml collecting pipes, and lid is opened, 13400rpm centrifugations 5min；Centrifugal column is placed in into new 1.5ml EP to manage
In, 25 μ l RNase-free water are added to the filter membrane in centrifugal column center, lid are closed, at a temperature of 15~25 DEG C of room temperature
Stand 1min；13400rpm centrifuges 1min；The RNA of extraction carries out reverse transcription or frozen in -80 DEG C immediately；
(6) RNA QC are assessed：RNA is carried out using Nanodrop quantitative and refer to A260/280 data, or used
Labchip/2100 carries out RNA quality evaluations, assesses RIN values.
The beneficial effects of the invention are as follows：The limitation based on detection technique in the prior art of the invention, using more increasing spirit
Quick ddPCR methods are detected, it is intended to have directive significance to clinic, its sensitivity is up to 0.01%.As shown in Fig. 2 this
The conception for inventing detection is as follows：The present invention is directed to common Brc3 types (accounting for 5%), design detection fusion probes (FAM), draws
Thing is expanded for fusion both sides sequence；Using RAR Α genes as internal reference, design Ref probes (HEX), primer only expands
RAR Α genes.The present invention identifies that the advantages of Brc3 hypotypes in PML-RAR alpha fusion genes has using ddPCR methods：1、ddPCR
PML-RAR Α fusion bcr3 hypotypes are detected up to the 0.1% of 0.01% abundance, far super RT-PCR, reproducible, no vacation is positive
Property；2nd, in the case of low AF (0.01%), it is proposed that the increase of sample input amount, or repeat multiple operation repetitives.Due to the technology
High sensitivity, therefore the early stage that can be applied to disease make a definite diagnosis, dynamic monitoring and preventing and treating recurrence.
Brief description of the drawings
Fig. 1 is PML-RAR α configuration pictures of the present invention；
Fig. 2 is Brc3 configuration pictures of the present invention；
Fig. 3 is ddPCR of the present invention result column diagram.
Embodiment
Below in conjunction with the drawings and specific embodiments, the invention will be further described.
A kind of Brc3 hypotypes identified using ddPCR methods in PML-RAR alpha fusion genes, the ddPCR methods are included
Extract the RNA in blood and QC is assessed, specific operation process is as follows：
(1) RNA is extracted, blood plasma is divided in 1.5ml EP pipes, often pipe dispenses 300 μ l；Using in Tiangeng kit from
The cell pyrolysis liquid CL of band, 750 μ l are added into blood, mixing of turning upside down, stand 1min；10000rpm centrifuges 1min；Abandon
Supernatant, stay precipitation；700 μ l CL are added into blood, is centrifuged under equal conditions, abandons supernatant, stay precipitation；
(2) 150 μ l buffer PKD are added, piping and druming mixes；Then 10 μ l Proteinase Ks are added, piping and druming mixes；Sample in
3min on ice is immediately placed in after 56 DEG C of thermostat, rotating speed 350rpm, 15min；13400rpm centrifuges 15min, transfer supernatant 160 μ l
Into 1.5ml EP pipes；Supernatant is placed in 80 DEG C of incubations 15min, 13400rpm on thermostat and quickly centrifuges 30s；Add 320 μ l
Buffer RLT, piping and druming mix, and add the ethanol of 1000 μ l 96~100%, are vortexed and mix；
(3) 700 μ l samples of transfer into RNeasy MinElute centrifugal columns, 13400rpm centrifugation 15s, abandon filtrate every time,
This step is repeated to finish until all samples are crossed into post；350 μ l buffer FRN are added to centrifuge 15s to centrifugal column, 13400rpm,
Abandon filtrate；
(4) mix is matched somebody with somebody:10 μ l DNaseI and 70 μ l buffer RDD, piping and druming mix, the film being added into centrifugal column
On, it is placed in 15min at a temperature of 20~30 DEG C of room temperature；500 μ l buffer FRN are added 13400rpm centrifugation 15s, to be protected to centrifugal column
Reserved filtrate；A new 2ml collecting pipe is changed, filtrate is rejoined into centrifugal column, 13400rpm centrifugation 15s, abandons filtrate；
(5) 500 μ l buffer RPE are added to centrifugal column, 13400rpm centrifugation 15s, filtrate is abandoned, is repeated once；Will be from
Stem is placed in new 2ml collecting pipes, and lid is opened, 13400rpm centrifugations 5min；Centrifugal column is placed in into new 1.5ml EP to manage
In, 25 μ l RNase-free water are added to the filter membrane in centrifugal column center, lid are closed, at a temperature of 15~25 DEG C of room temperature
Stand 1min；13400rpm centrifuges 1min；The RNA of extraction carries out reverse transcription or frozen in -80 DEG C immediately；
(6) RNA QC are assessed：RNA is carried out using Nanodrop quantitative and refer to A260/280 data, or used
Labchip/2100 carries out RNA quality evaluations, assesses RIN values.
DdPCR methods in the present invention also include RNA reverse transcriptions cDNA and carry out QC assessments, and specific operation process is as follows：
(1) RNA reverse transcriptions cDNA, reverse transcription primer are combined with template ribonucleic acid, after PCR EPs (end of program), are positioned on ice,
No less than 1min；HELA need to only put into 1ul, i.e. 10ng as control.
(2) prepare RT reaction mixtures and carry out RT incubations：7ul mix are added into the 13ul of previous step, piping and druming is mixed
It is even, total system 20ul is reacted, reverse transcription reaction is carried out on PCR, storage extremely -20 DEG C of preservations after the completion of reaction.
(3) cDNA QC are assessed：The HELA control primer carried using kit, enter performing PCR reaction, run glue
Verify band, the performance of Quality Control reverse transcription reaction.
DdPCR systems：
DdPCR programs：
·Use a heated lid set to 105℃ and set the sample volume to 40 μl.
Embodiment：Blood sample and NB4- simulations AF 0.01%.
RNA extracts result --- Nanodrop
| Sample names | Concentration (ng/ul) | Total amount (ug) | A260/280 | A260/230 |
| WBC | 105 | 3ug | 2.07 | 1.10 |
| NB4 | 790.2 | 15ug | 2.11 | 1.98 |
DdPCR result column diagram is as shown in Figure 3.First row：Without false sun detection, postive numbers are 0.Secondary series：
K562 pattern detections, high abundance sample.3rd row to the 5th row：For 0.05% sample, its input amount gradually increases AF,
Positive detections number also increases.6th row to the 8th row：For 0.01% sample, its input amount gradually increases AF,
Positive detections number also increases.
Finally it should be noted that above content is merely illustrative of the technical solution of the present invention, rather than the present invention is protected
The limitation of scope, the simple modification or equivalent substitution that one of ordinary skill in the art is carried out to technical scheme,
All without departing from the spirit and scope of technical solution of the present invention.
Claims (1)
- A kind of 1. Brc3 hypotypes identified using ddPCR methods in PML-RAR alpha fusion genes, it is characterised in that：The ddPCR Method is assessed including the RNA in extraction blood and QC, and specific operation process is as follows：（1）RNA is extracted, blood plasma is divided in 1.5ml EP pipes, often pipe dispenses 300 μ l；Use what is carried in Tiangeng kit Cell pyrolysis liquid CL, 750 μ l are added into blood, mixing of turning upside down, stand 1min；10000rpm centrifuges 1min；Abandon supernatant, Stay precipitation；700 μ l CL are added into blood, is centrifuged under equal conditions, abandons supernatant, stay precipitation；（2）150 μ l buffer PKD are added, piping and druming mixes；Then 10 μ l Proteinase Ks are added, piping and druming mixes；Sample is in constant temperature 3min on ice is immediately placed in after 56 DEG C of instrument, rotating speed 350rpm, 15min；13400rpm centrifuges 15min, and transfer supernatant 160 μ l are extremely In 1.5ml EP pipes；Supernatant is placed in 80 DEG C of incubations 15min, 13400rpm on thermostat and quickly centrifuges 30s；Add 320 μ l Buffer RLT, piping and druming mix, and add the ethanol of 1000 μ l 96~100%, are vortexed and mix；（3）700 μ l samples of transfer 13400rpm centrifugation 15s, are abandoned filtrate, repeated into RNeasy MinElute centrifugal columns every time This step finishes until all samples are crossed into post；350 μ l buffer FRN are added 13400rpm centrifugation 15s, to abandon filter to centrifugal column Liquid；（4）With mix:10 μ l DNaseI and 70 μ l buffer RDD, piping and druming mix, are added on the film in centrifugal column, put The 15min at a temperature of 20~30 DEG C of room temperature；500 μ l buffer FRN are added 13400 rpm centrifugation 15s, to retain filter to centrifugal column Liquid；A new 2ml collecting pipe is changed, filtrate is rejoined into centrifugal column, 13400rpm centrifugation 15s, abandons filtrate；（5）500 μ l buffer RPE are added to centrifugal column, 13400rpm centrifugation 15s, filtrate is abandoned, is repeated once；By centrifugal column It is placed in new 2ml collecting pipes, lid is opened, 13400rpm centrifugations 5min；Centrifugal column is placed in new 1.5ml EP pipes, added Enter 25 μ l RNase-free water to the filter membrane in centrifugal column center, close lid, stood at a temperature of 15~25 DEG C of room temperature 1min；13400rpm centrifuges 1min；The RNA of extraction carries out reverse transcription or frozen in -80 DEG C immediately；（6）RNA QC are assessed：RNA is carried out using Nanodrop quantitative and refer to A260/280 data, or use labchip / 2100 carry out RNA quality evaluations, assess RIN values.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710505991.2A CN107523615A (en) | 2017-06-28 | 2017-06-28 | Brc3 hypotypes in PML RAR alpha fusion genes are identified using ddPCR methods |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710505991.2A CN107523615A (en) | 2017-06-28 | 2017-06-28 | Brc3 hypotypes in PML RAR alpha fusion genes are identified using ddPCR methods |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN107523615A true CN107523615A (en) | 2017-12-29 |
Family
ID=60748232
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201710505991.2A Pending CN107523615A (en) | 2017-06-28 | 2017-06-28 | Brc3 hypotypes in PML RAR alpha fusion genes are identified using ddPCR methods |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN107523615A (en) |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102719540A (en) * | 2012-06-12 | 2012-10-10 | 福州艾迪康医学检验所有限公司 | Detection kit for detecting relative expression level of PML-RAR (alpha) fusion gene by use of fluorescent quantitative PCR (polymerase chain reaction) technology |
| CN105229175A (en) * | 2013-03-15 | 2016-01-06 | 雅培分子公司 | For increasing and measuring the method for RNA fusion gene variant, the method distinguishing them and relevant primer, probe and test kit |
-
2017
- 2017-06-28 CN CN201710505991.2A patent/CN107523615A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102719540A (en) * | 2012-06-12 | 2012-10-10 | 福州艾迪康医学检验所有限公司 | Detection kit for detecting relative expression level of PML-RAR (alpha) fusion gene by use of fluorescent quantitative PCR (polymerase chain reaction) technology |
| CN105229175A (en) * | 2013-03-15 | 2016-01-06 | 雅培分子公司 | For increasing and measuring the method for RNA fusion gene variant, the method distinguishing them and relevant primer, probe and test kit |
Non-Patent Citations (5)
| Title |
|---|
| CLAUDIA BRUNETTI等: "Droplet digital PCR is a reliable tool for monitoring minimal residual disease in acute promyelocytic leukemia", 《THE JOURNAL OF MOLECULAR DIAGNOSTICS》 * |
| FRANCESCO ALBANO等: "Absolute quantification of the pretreatment PML-RARA transcript defines the relapse risk in acute promyelocytic leukemia", 《ONCOTARGET》 * |
| 刘涛: "《大型海藻实验技术》", 31 May 2016, 海洋出版社 * |
| 卢大儒等: "《生物"芯"时代》", 30 April 2005, 上海科学普及出版社 * |
| 韩兰秀等: "建立RQ-PCR方法检测急性早幼粒细胞白血病患者6种不同PML/RARα异构体", 《江苏大学学报(医学版)》 * |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| JP7358434B2 (en) | Urine Biomarker Cohorts, Gene Expression Characteristics, and Methods of Their Use | |
| Egidi et al. | Stability assessment of candidate reference genes in urine sediment of prostate cancer patients for miRNA applications | |
| Alvarez | Isolation of urinary exosomes for RNA biomarker discovery using a simple, fast, and highly scalable method | |
| CN106148495A (en) | The application in colorectal cancer biomarker of a kind of circular rna | |
| CN102807976B (en) | Rapid nucleic acid extraction kit and applications thereof | |
| CN105112564A (en) | Method and kit for detecting high-risk HPV (human papillomavirus) E6/E7 mRNA (messenger ribonucleic acid) by ligase | |
| Leong et al. | based microRNA expression profiling from plasma and circulating tumor cells | |
| CN107529556A (en) | A kind of method that Brc2 hypotypes in PML RAR alpha fusion genes are identified using ddPCR | |
| CN107523615A (en) | Brc3 hypotypes in PML RAR alpha fusion genes are identified using ddPCR methods | |
| CN105506156A (en) | Molecular marker for diagnosing osteosarcoma | |
| CN105441565A (en) | miRNA regarding as osteosarcoma treatment target | |
| CN105256068A (en) | Novel high-sensitivity respiratory virus nucleic acid NASBA (nucleic acid sequence based amplification) primers and detection method | |
| CN111321224B (en) | miRNA biomarker combination for diagnosis or auxiliary diagnosis of gastric cancer and kit thereof | |
| CN101921743A (en) | Method for acquiring genetic information of ancient animal | |
| CN107529557A (en) | The method that Brc1 hypotypes in PML RAR alpha fusion genes are identified using ddPCR | |
| CN109536502B (en) | PCR (polymerase chain reaction) internal reference applicable to plasma exosome miRNA of patient with gestational trophoblastic tumor | |
| CN106148494A (en) | The application in colorectal cancer biomarker of a kind of circular rna | |
| CN105779640A (en) | miRNA biomarker and detection kit used for renal cancer diagnosis | |
| CN109136382B (en) | Method and system for identifying four human body fluid sources | |
| CN105779606B (en) | The marker of diagnosis and indication focal segmental glomerulosclerosis | |
| Sura et al. | Molecular testing opportunities on cytology effusion specimens: the pre-analytic effects of various body fluid cytology preparation methods on RNA extraction quality and targeted sequencing | |
| Somura et al. | A three-gene predictor for early intrahepatic recurrence of hepatocellular carcinoma after curative hepatectomy | |
| CN103789447A (en) | Method for detecting 5'end tRNA semi-molecules | |
| Cho-Vega et al. | Combined laser capture microdissection and serial analysis of gene expression from human tissue samples | |
| CN114517233A (en) | Primer probe combination for early warning and clinical diagnosis of colorectal cancer |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| RJ01 | Rejection of invention patent application after publication | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20171229 |