CN107529557A - The method that Brc1 hypotypes in PML RAR alpha fusion genes are identified using ddPCR - Google Patents
The method that Brc1 hypotypes in PML RAR alpha fusion genes are identified using ddPCR Download PDFInfo
- Publication number
- CN107529557A CN107529557A CN201710505924.0A CN201710505924A CN107529557A CN 107529557 A CN107529557 A CN 107529557A CN 201710505924 A CN201710505924 A CN 201710505924A CN 107529557 A CN107529557 A CN 107529557A
- Authority
- CN
- China
- Prior art keywords
- rna
- added
- 13400rpm
- ddpcr
- centrifugal column
- 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
Landscapes
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
- Investigating Or Analysing Biological Materials (AREA)
Abstract
The invention discloses a kind of method that Brc1 hypotypes in PML RAR alpha fusion genes are identified using ddPCR, this method includes the RNA in extraction blood and carries out QC assessments, in addition to RNA reverse transcriptions cDNA and carries out QC assessments.The present invention is detected using more highly sensitive ddPCR methods, it is intended to has directive significance to clinic, its sensitivity is up to 0.01%.DdPCR detection methods PML RAR α of the present invention fusion bcr1 hypotypes up to 0.01% abundance, the 0.1% of remote super RT PCR, reproducible, non-false positive；In low AF（0.01%）In the case of, 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 ddPCR identifications PML-
The method of Brc1 hypotypes in 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 (brc2, 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：Elongated (L-type, brc1), anomaly (V-type, brc2) and short (S types, brc3), as shown in Figure 1.
The treatment of leukaemia is more effective at present, and its patient is extended life cycle, but later stage follow-up is found in the several years again
Hair rate is higher, and possible cause is its detection sensitivity is only 0.1% using RT-PCR detection techniques at present.Based on the detection skill
The limitation of art, this project are detected using more highly sensitive ddPCR methods, it is intended to there is directive significance to clinic, its
Sensitivity is up to 0.01%.The conception of this detection is as follows：For most commonly seen brc1 types (accounting for 55%), design detection
Fusion probes (FAM), primer are expanded for fusion both sides sequence；Visited RAR Α genes as internal reference, design Ref
Pin (HEX), primer only expand RAR Α genes, as shown in Figure 2.
The content of the invention
The present invention is in order to overcome the shortcomings of the prior art, there is provided one kind has highly sensitive to be identified using ddPCR
The method of Brc1 hypotypes in PML-RAR alpha fusion genes.
The present invention is achieved by the following technical solutions：One kind is using in ddPCR identification PML-RAR alpha fusion genes
The method of Brc1 hypotypes, this method include the RNA in extraction blood and carry out QC assessments, and specific operation process is as follows：
Extract RNA：
(1) blood plasma is divided in 1.5ml EP pipes, often pipe dispenses 300 μ l；
(2) using the cell pyrolysis liquid CL carried in Tiangeng kit, 750 μ l are added into blood, mixing of turning upside down,
Stand 1min；10000rpm centrifuges 1min；Supernatant is abandoned, stays precipitation；700 μ l CL are added into blood, under equal conditions from
The heart, supernatant is abandoned, stay precipitation；
(3) 150 μ l buffer PKD are added, piping and druming mixes；Then 10 μ l Proteinase Ks are added, piping and druming mixes；Do not configure
Mix, need to individually it sequentially add；
(4) sample is immediately placed in 3min on ice after 56 DEG C of thermostat, rotating speed 350rpm, 15min；
(5) 13400rpm centrifuges 15min, shifts in μ l to the 1.5ml EP pipes of supernatant 160；
(6) supernatant is placed in 80 DEG C of incubations 15min, 13400rpm on thermostat and quickly centrifuges 30s；
(7) 320 μ l buffer RLT are added, piping and druming mixes, and adds the ethanol of 1000 μ l 96~100%, is vortexed and mixes；
(8) 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；
(9) plus 350 μ l buffer FRN are to centrifugal column, 13400rpm centrifugation 15s, abandon filtrate；
(10) 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；
(11) plus 500 μ l buffer FRN are to centrifugal column, 13400rpm centrifugation 15s, retain filtrate；Change a new 2ml
Collecting pipe, filtrate is rejoined into centrifugal column, 13400rpm centrifugation 15s, abandons filtrate；
(12) 500 μ l buffer RPE are added to centrifugal column, 13400rpm centrifugation 15s, filtrate is abandoned, is repeated once；
(13) centrifugal column is placed in new 2ml collecting pipes, lid is opened, 13400rpm centrifugations 5min；
(14) centrifugal column is placed in new 1.5ml EP pipes, adds 25 μ l RNase-free water into centrifugal column
On the filter membrane of centre, lid is closed, 1min is stood at a temperature of 15~25 DEG C of room temperature；13400rpm centrifuges 1min；
(15) RNA of extraction carries out reverse transcription or frozen in -80 DEG C immediately；
RNA QC are assessed：
(1) RNA is carried out using Nanodrop quantitative and refers to A260/280 data；
(2) RNA quality evaluations are carried out using labchip/2100, assesses RIN values.
RNA reverse transcriptions cDNA：
(1) reverse transcription primer is combined with template ribonucleic acid：
After PCR EPs (end of program), it is 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 mixes, and reacts total system 20ul, is carried out on PCR anti-
Responsive transcription, 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 checking band, Quality Control reversion
Record the performance of reaction.
DdPCR systems：
DdPCR programs：
-Use a heated lid set to 105℃ and set the sample volume to 40μl.
The beneficial effects of the invention are as follows：The present invention identifies the side of Brc1 hypotypes in PML-RAR alpha fusion genes using ddPCR
The advantages of method, has：1st, ddPCR detects PML-RAR Α fusion bcrl hypotypes up to 0.01% abundance, remote super RT-PCR's
0.1%, reproducible, non-false positive；2nd, in the case of low AF (0.01%), it is proposed that the increase of sample input amount, or repeat more
Individual operation repetitive.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 answered
Hair.
Brief description of the drawings
Fig. 1 is PML-RAR α configuration pictures of the present invention；
Fig. 2 is brc1 configuration pictures of the present invention；
Fig. 3 is cDNA of the present invention QC result figures：
Fig. 4 is ddPCR of the present invention result column diagram；
Fig. 5 is RNA extractions result figure --- Labchip of the present invention.
Embodiment
Below in conjunction with the drawings and specific embodiments, the invention will be further described.
A kind of method that Brc1 hypotypes in PML-RAR alpha fusion genes are identified using ddPCR, this method include extraction blood
In RNA and carry out QC assessments, specific operation process is as follows：
Extract RNA：
(1) blood plasma is divided in 1.5ml EP pipes, often pipe dispenses 300 μ l；
(2) using the cell pyrolysis liquid CL carried in Tiangeng kit, 750 μ l are added into blood, mixing of turning upside down,
Stand 1min；10000rpm centrifuges 1min；Supernatant is abandoned, stays precipitation；700 μ l CL are added into blood, under equal conditions from
The heart, supernatant is abandoned, stay precipitation；
(3) 150 μ l buffer PKD are added, piping and druming mixes；Then 10 μ l Proteinase Ks are added, piping and druming mixes；Do not configure
Mix, need to individually it sequentially add；
(4) sample is immediately placed in 3min on ice after 56 DEG C of thermostat, rotating speed 350rpm, 15min；
(5) 13400rpm centrifuges 15min, shifts in μ l to the 1.5ml EP pipes of supernatant 160；
(6) supernatant is placed in 80 DEG C of incubations 15min, 13400rpm on thermostat and quickly centrifuges 30s；
(7) 320 μ l buffer RLT are added, piping and druming mixes, and adds the ethanol of 1000 μ l 96~100%, is vortexed and mixes；
(8) 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；
(9) plus 350 μ l buffer FRN are to centrifugal column, 13400rpm centrifugation 15s, abandon filtrate；
(10) 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；
(11) plus 500 μ l buffer FRN are to centrifugal column, 13400rpm centrifugation 15s, retain filtrate；Change a new 2ml
Collecting pipe, filtrate is rejoined into centrifugal column, 13400rpm centrifugation 15s, abandons filtrate；
(12) 500 μ l buffer RPE are added to centrifugal column, 13400rpm centrifugation 15s, filtrate is abandoned, is repeated once；
(13) centrifugal column is placed in new 2ml collecting pipes, lid is opened, 13400rpm centrifugations 5min；
(14) centrifugal column is placed in new 1.5ml EP pipes, adds 25 μ l RNase-free water into centrifugal column
On the filter membrane of centre, lid is closed, 1min is stood at a temperature of 15~25 DEG C of room temperature；13400rpm centrifuges 1min；
(15) RNA of extraction carries out reverse transcription or frozen in -80 DEG C immediately；
RNA QC are assessed：
(1) RNA is carried out using Nanodrop quantitative and refers to A260/280 data；
(2) RNA quality evaluations or using labchip/2100 are carried out, assess RIN values.
This method also includes RNA reverse transcriptions cDNA and carries out QC assessments, and specific operation process is as follows：
RNA reverse transcriptions cDNA：
(1) reverse transcription primer is combined with template ribonucleic acid；
After PCR EPs (end of program), it is positioned on ice, no less than 1min；HELA need to only put into 1ul as control, i.e.,
10ng；
(2) prepare RT reaction mixtures and carry out RT incubations；
7ul mix are added into the 13ul of previous step, piping and druming mixes, and reacts total system 20ul, is carried out on PCR anti-
Responsive transcription, storage extremely -20 DEG C of preservations after the completion of reaction；
CDNA QC are assessed：The HELA control primer carried using kit, enter performing PCR reaction, run glue checking
Band, the performance of Quality Control reverse transcription reaction.
Embodiment：Blood sample and NB4- simulations AF 0.01%.
RNA is extracted and reverse transcription reagent box：
《TIANGEN TIANamp Blood DNA Kit poba gene group DNA extraction kits》
《QIAGEN ALLPrep DNA/RNA FFPE Kit》
《Invitrogen cDNA kits》
Table one：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 |
CDNA QC results are as shown in Figure 3.DdPCR result is as shown in Figure 4.
First row：Without false sun detection, postive numbers are 0.Secondary series：K562 pattern detections, high abundance sample.3rd
Arrange to the 5th row：For 0.05% sample, its input amount gradually increases AF, and positive detections number also increases.6th row are extremely
8th row：For 0.01% sample, its input amount gradually increases AF, and 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 (2)
- A kind of 1. method that Brc1 hypotypes in PML-RAR alpha fusion genes are identified using ddPCR, it is characterised in that：This method includes Extract the RNA in blood and carry out QC assessments, specific operation process is as follows：Extract RNA：（1）Blood plasma is divided in 1.5ml EP pipes, often pipe dispenses 300 μ l；（2）Using the cell pyrolysis liquid CL carried in Tiangeng kit, 750 μ l are added into blood, mixing of turning upside down, are stood 1min；10000rpm centrifuges 1min；Supernatant is abandoned, stays precipitation；700 μ l CL are added into blood, centrifuges, abandons under equal conditions Supernatant, stay precipitation；（3）150 μ l buffer PKD are added, piping and druming mixes；Then 10 μ l Proteinase Ks are added, piping and druming mixes；Mix is not configured, Need to individually it sequentially add；（4）Sample is immediately placed in 3min on ice after 56 DEG C of thermostat, rotating speed 350rpm, 15min；（5）13400rpm centrifuges 15min, shifts in μ l to the 1.5ml EP pipes of supernatant 160；（6）Supernatant is placed in 80 DEG C of incubations 15min, 13400rpm on thermostat and quickly centrifuges 30s；（7）320 μ l buffer RLT are added, piping and druming mixes, and adds the ethanol of 1000 μ l 96~100%, is vortexed and mixes；（8）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；（9）350 μ l buffer FRN are added 13400rpm centrifugation 15s, to abandon filtrate to centrifugal column；（10）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；（11）500 μ l buffer FRN are added 13400 rpm centrifugation 15s, to retain filtrate to centrifugal column；A new 2ml is changed to receive Collector, filtrate is rejoined into centrifugal column, 13400rpm centrifugation 15s, abandons filtrate；（12）500 μ l buffer RPE are added to centrifugal column, 13400rpm centrifugation 15s, filtrate is abandoned, is repeated once；（13）Centrifugal column is placed in new 2ml collecting pipes, lid is opened, 13400rpm centrifugations 5min；（14）Centrifugal column is placed in new 1.5ml EP pipes, adds 25 μ l RNase-free water to centrifugal column center On filter membrane, lid is closed, 1min is stood at a temperature of 15~25 DEG C of room temperature；13400rpm centrifuges 1min；（15）The RNA of extraction carries out reverse transcription or frozen in -80 DEG C immediately；RNA QC are assessed：（1）RNA is carried out using Nanodrop quantitative and refer to A260/280 data；（2）RNA quality evaluations are carried out using labchip/2100, assess RIN values.
- 2. the method according to claim 1 that Brc1 hypotypes in PML-RAR alpha fusion genes are identified using ddPCR, its feature It is：This method also includes RNA reverse transcriptions cDNA and carries out QC assessments, and specific operation process is as follows：RNA reverse transcriptions cDNA：（1）Reverse transcription primer is combined with template ribonucleic acid；After PCR EPs (end of program), it is positioned on ice, no less than 1 min；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, and reacts total system 20ul, and it is anti-that reverse transcription is carried out on PCR Should, storage extremely -20 DEG C of preservations after the completion of reaction；CDNA QC are assessed：The HELA control primer carried using kit, enter performing PCR reaction, run glue checking band, The performance of Quality Control reverse transcription reaction.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710505924.0A CN107529557A (en) | 2017-06-28 | 2017-06-28 | The method that Brc1 hypotypes in PML RAR alpha fusion genes are identified using ddPCR |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710505924.0A CN107529557A (en) | 2017-06-28 | 2017-06-28 | The method that Brc1 hypotypes in PML RAR alpha fusion genes are identified using ddPCR |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN107529557A true CN107529557A (en) | 2018-01-02 |
Family
ID=60766371
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201710505924.0A Pending CN107529557A (en) | 2017-06-28 | 2017-06-28 | The method that Brc1 hypotypes in PML RAR alpha fusion genes are identified using ddPCR |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN107529557A (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 CN201710505924.0A patent/CN107529557A/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 |
|---|---|---|
| William et al. | Genomic identification of direct target genes of LEAFY | |
| Nuovo | In situ detection of precursor and mature microRNAs in paraffin embedded, formalin fixed tissues and cell preparations | |
| CN110191962A (en) | The sequencing and analysis of allochthon associated nucleic acid | |
| CN112251492A (en) | Kit and method for extracting virus nucleic acid | |
| CN102807976B (en) | Rapid nucleic acid extraction kit and applications thereof | |
| WO2016023431A1 (en) | Wash solution and washing method for hybrid-enrichment-capture dna sequencing library | |
| CN113999909A (en) | Serum exosome marker for gastric cancer diagnosis, application thereof, amplification primer pair and diagnosis kit | |
| Macedo et al. | Maximizing total RNA yield from TRIzol reagent protocol: a feasibility study | |
| CN103789197B (en) | A kind of test kit and extracting method thereof extracting Microrna | |
| CN103361346A (en) | Method for cloning and analyzing populus diversifolia micro RNAs (ribonucleic acids) precursor | |
| CN107541550A (en) | People's ROS1 fusion detection methods | |
| CN107529556A (en) | A kind of method that Brc2 hypotypes in PML RAR alpha fusion genes are identified using ddPCR | |
| CN107529557A (en) | The method that Brc1 hypotypes in PML RAR alpha fusion genes are identified using ddPCR | |
| CN101921743A (en) | Method for acquiring genetic information of ancient animal | |
| CN110205365B (en) | High-throughput sequencing method for efficiently researching RNA interaction group and application thereof | |
| CN101914523B (en) | Method for separating RNA (Ribonucleic Acid) from human serum/blood plasma sample and PCR (Polymerase Chain Reaction) verification method thereof | |
| CN107523615A (en) | Brc3 hypotypes in PML RAR alpha fusion genes are identified using ddPCR methods | |
| CN114525274A (en) | Lysate and centrifugation column method virus nucleic acid extraction kit based on lysate | |
| CN104630222B (en) | A kind of miRNA and the specific primer being detected to it and method | |
| CN107058539A (en) | Reference gene and its application for barley gene expression study | |
| CN102181435A (en) | Method for quickly extracting ribonucleic acid (RNA) from ginseng leaf tissues | |
| CN108728528B (en) | Fluorescent quantitative PCR one-step kit for detecting specific gene of renal tubular epithelial cell based on Taqman probe | |
| US11021756B2 (en) | MiRNA markers for the diagnosis of osteosarcoma | |
| CN103952399A (en) | Method of extracting ribonucleic acid by using phenol guanidine salt lysate containing indicator | |
| CN101220390B (en) | Method for rapidly extracting plants sample DNA |
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 |
Application publication date: 20180102 |
|
| RJ01 | Rejection of invention patent application after publication |