CN107345249A - Applications of the hsa_circRNA_103112 in the diagnosis, treatment and prognosis of Down syndrome - Google Patents
Applications of the hsa_circRNA_103112 in the diagnosis, treatment and prognosis of Down syndrome Download PDFInfo
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- CN107345249A CN107345249A CN201710493867.9A CN201710493867A CN107345249A CN 107345249 A CN107345249 A CN 107345249A CN 201710493867 A CN201710493867 A CN 201710493867A CN 107345249 A CN107345249 A CN 107345249A
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Abstract
The invention discloses a kind of CircRNA, its sequence such as SEQ ID NO：Shown in 1；And further disclose the primer for the RT PCR for detecting the CircRNA.CircRNA expression in Down syndrome patient and normal population body has obvious differential expression, clinical assistant diagnosis technology currently used for Down syndrome is not perfect, therefore the CircRNA has the potential of the biomarker related as Down syndrome.
Description
Technical field
The invention belongs to biological technical field, more particularly to a kind of new ring-type hsa_circRNA_103112 and its should
With.
Background technology
Down syndrome (Down Syndrome, DS), i.e., 21- patau syndromes, also referred to as mongolism or Down are comprehensive
Simulator sickness, it is due to inborn defect class disease caused by No. 21 chromosome abnormality, is most common one in birth defect
Kind chromosome aneuploid genetic disease.Pathogenesis based on Down syndrome is still unclear, currently for the disease still
There is no effective treatment method, it is the current important means for reducing inborn defect that birth, which is intervened, and intervention of being born depends on
Pre-natal diagnosis and Prenatal Screening.Prenatal Screening is mainly in (15~20 weeks) detection pregnant woman of pregnant early stage (7~12 weeks) and second trimester
Examination, Ultrasonic screening, the serological examination of fetomaternal and United screening scheme of certain index including pregnant woman age etc.
To assess the risk whether fetus suffers from DS.Traditional pre-natal diagnosis then mainly using invasive sampling means, including
Umbilical cord takes blood, amniocentesis and chorionic villous sampling etc., utilizes DS genetic diagnosis technology, DS Molecular diagnosis technology, glimmering
The technologies such as light in situ hybridization (FISH), PCR (QF-PCR) and STR (STR) are entered to fetus sample
Row analyzing and diagnosing.However, traditional Prenatal Screening has, recall rate is low, loss is high and lacking for false positive easily occurs in result
Point, pre-natal diagnosis is then because the means of intrusive mood sampling easily produce bleeding, miscarriage or with other complication.
In order to improve the security of the accuracy of Prenatal Screening and pre-natal diagnosis, researchers are exploring new DS always
Non-invasive methods for prenatal diagnosis.Non-invasive Prenatal Diagnosis have the advantages that it is easy, safe and reliable, including female blood tire
Youngster's cellular pathways, female blood fetus dissociative DNA approach and female blood fetus dissociative RNA approach.Yet with fetal nucleated cell
Genome covers the whole hereditary information of fetus, thus the fetal nucleated cell being present in pregnant woman's circulating becomes initially
The research direction of researchers.But greatest problem existing for this method in fetal nucleated cell in maternal blood content pole
It is few, the requirement of analyzing and diagnosing is unable to reach, clinical practice is received certain limitation.
In summary, based on traditional Prenatal Screening and the defects of pre-natal diagnosis, and existing Non-invasive Prenatal Diagnosis method
Deficiency so that explore new non-invasive Down syndrome fetus pre-natal diagnosis mark and have very important significance.
The content of the invention
It is an object of the invention to disclose a kind of new ring-type hsa_circRNA_103112.
The technical solution used in the present invention is：A kind of circular rna is disclosed, its sequence such as SEQ ID NO：Shown in 1.
Preferably, the circular rna is as Down syndrome detection, treatment, the application of prognosis target spot.
A kind of Down syndrome detection kit, containing detection sequence can be quantified it is SEQ ID NO in the kit：1
Circular rna reagent.
Preferably, it is SEQ ID NO containing real time fluorescent quantitative detection sequence in the kit：The primer of 1 circular rna
Sequence.
Preferably, detection circular rna primer sequence is：
F：5’-GCACTTCCTGGCAATGATAGAT-3’(SEQ ID NO：4)；
R：5’-GGCTTGCTGTAGTATCTGGGTG-3’(SEQ ID NO：5).
Quantitative detection sequence is SEQ ID NO：The reagent of 1 circular rna is in Down syndrome detection reagent is prepared
Using.
Preferably, quantitative detection sequence such as SEQ ID NO：The reagent of circular rna shown in 1 is that real time fluorescent quantitative detects sequence
Row such as SEQ ID NO：The primer sequence of circular rna shown in 1.
It is further preferred that circular rna primer sequence is：
F：5’-GCACTTCCTGGCAATGATAGAT-3’(SEQ ID NO：4)；
R：5’-GGCTTGCTGTAGTATCTGGGTG-3’(SEQ ID NO：5).
The beneficial effects of the invention are as follows：CircRNA disclosed in the present patent application is in Down syndrome patient and normal population
Expression has obvious differential expression in vivo, and the clinical assistant diagnosis technology currently used for Down syndrome is not perfect,
Therefore the CircRNA has the potential of the biomarker related as Down syndrome.
Embodiment
Embodiment 1
In the research process to Down's syndrome, a kind of circular rna is found, its sequence is：
CACCAGCAGACGTTTTTGAATCAACTGAGAGAAATTACGGGGATTAATGACACCCAGAT
ACTACAGCAAGCCTTGAAGGATAGTAATGGAAACTTGGAATTAGCAGTGGCTTTCCTTA
CTGCGAAGAATGCTAAGACCCCTCAGCAGGAGGAGACAACTTACTACCAAACAGCACT
TCCTGGCAATGATAGATACATCAGTGTGGGAAGCCAAGCAGATACAA(SEQ ID NO：1) hsa_, is named
circRNA_103112。
The sample of embodiment 2 and processing
12 fetal cord blood specimens obtain from Shenzhen people's hospital clinic study revenue centre laboratory, wherein
Including 6 through G banding chromosome karyotypings be diagnosed as the umbilical cord blood specimen (2 male 4 female) of standard type Down syndrome fetus with
And 6 normal fetus Cord bloods (2 male 4 female), pregnant age is 18-22 weeks；12 children peripheral blood specimens are from Guilin City Chinese
People PLA the first 81 Army Hospital Guangxi metabolic disease research emphasis laboratory obtains, and is integrated including 6 Tang Shi
Infant periphery blood specimen (2 male 4 female) and 6 healthy children periphery blood specimens (2 male 4 female) are levied, the age is 0-15 year.Originally grind
Study carefully the approval of Shenzhen people's hospital and Ethics Committee of Army Hospital of PLA of Guilin City first 81, and obtain
The informed consent of patient, all subjects are provided which Written informed consent.
RT-PCR technology is respectively adopted to be verified in the peripheral blood of DS infants and healthy children.Simultaneously for checking
MRNA corresponding to the circRNA of the otherness change drawn, inventor are examined using RT-PCR technology in peripheral blood
Survey.
RT-PCR checking specific experiment step be：
(1) the gradient dilution DNA profiling for drawing standard curve is prepared：
A selects a cDNA template for determining to express the gene to enter for each gene and house-keeping gene for needing to measure
Performing PCR reacts：
Ttom of pipe is flicked, solution is mixed, the of short duration centrifugations of 5000rpm, sets PCR to react：95 DEG C, 10min；40 PCR are followed
(95 DEG C, 10 seconds of ring；60 DEG C, 60 seconds (collection fluorescence))；
B is by PCR primer and 100bp DNA Ladder in 2% agarose gel electrophoresis, ethidium bromide staining, detection PCR
Whether product is single specificity amplified band；
PCR primer is carried out 10 times of gradient dilutions by c：PCR primer concentration is set as 1, is diluted to 1 × 10 respectively-1、1×10-2、 1×10-3、1×10-4、1×10-5、1×10-6、1×10-7、1×10-8、1×10-9, the DNA of these gradient concentrations.
(2) RT-PCR reactions are carried out：
RT-PCR reaction systems are respectively configured in all cDNA samples by a.System configurations are as follows：
Ttom of pipe is flicked, solution is mixed, 5000rpm, of short duration centrifugation；
B is loaded：
8ul mixed liquors are added in each hole corresponding to 384-PCR plates, add corresponding 2 μ L cDNA.Carefully it is stained with
Sealing Film sealed membranes, and of short duration centrifugation mixing.Finally, ready PCR plate is placed on ice before PCR programs are set
On；
Above-mentioned 384-PCR plates are placed in the enterprising performing PCR of Realtime PCR instruments and reacted by c：
All indexs are carried out by following procedure：95 DEG C, 10min；40 (95 DEG C, 10 seconds of PCR cycles；60 DEG C, 60
Second (collection fluorescence)).In order to establish the melting curve of PCR primer, after amplified reaction terminates, by (95 DEG C, 10 seconds；60 DEG C, 60
Second；95 DEG C, 15 seconds)；And 99 DEG C (instrument carries out-Ramp Rate as 0.05 DEG C/sec automatically) are heated slowly to from 60 DEG C.
(3) result is with calculating：
The sample-adding amount of each sample is 2 μ L during RT-PCR, yet with by RNA concentration quantitatives error and RNA reverse transcriptions effect
The influence of rate error etc., its content of the cDNA of 2 μ L volumes of each sample is not fully identical, to correct this difference, uses pipe
Family gene β-actin (different sample room expression quantity substantially constants) are used as internal reference, with the value of sample testing gene divided by this sample
The value of internal reference, the ratio finally given are the testing gene relative amount of sample.
The experimental result of embodiment 3
Using β-actin as internal reference thing, above-mentioned circRNA is tested in peripheral blood with RT-PCR technology respectively
Card.PCR primer sequence such as table 1：
The RT-PCR primer list of table 1
Fold differences are calculated using 2- Δ Δ Ct methods, as a result such as table 2.
The circRNA the results of table 2
As a result show, compared to healthy population, in Down's syndrome patient, hsa_circRNA_103112 expression quantity
Can significantly it raise, there is obvious positively related relation for the two.
SEQUENCE LISTING
<110>No.181 Hospital, PLA
<120>Applications of the hsa_circRNA_103112 in the diagnosis, treatment and prognosis of Down syndrome
<130>
<160> 5
<170> PatentIn version 3.5
<210> 1
<211> 223
<212> DNA
<213> hsa_circRNA_103112
<400> 1
caccagcaga cgtttttgaa tcaactgaga gaaattacgg ggattaatga cacccagata 60
ctacagcaag ccttgaagga tagtaatgga aacttggaat tagcagtggc tttccttact 120
gcgaagaatg ctaagacccc tcagcaggag gagacaactt actaccaaac agcacttcct 180
ggcaatgata gatacatcag tgtgggaagc caagcagata caa 223
<210> 2
<211> 20
<212> DNA
<213>Artificial sequence
<400> 2
gtggccgagg actttgattg 20
<210> 3
<211> 23
<212> DNA
<213>Artificial sequence
<400> 3
cctgtaacaa cgcatctcat att 23
<210> 4
<211> 22
<212> DNA
<213>Artificial sequence
<400> 4
gcacttcctg gcaatgatag at 22
<210> 5
<211> 22
<212> DNA
<213>Artificial sequence
<400> 5
ggcttgctgt agtatctggg tg 22
Claims (8)
1. a kind of circular rna, its sequence are：
CACCAGCAGACGTTTTTGAATCAACTGAGAGAAATTACGGGGATTAATGACACCCAGATACTACAGCAAGCCT
TGAAGGATAGTAATGGAAACTTGGAATTAGCAGTGGCTTTCCTTACTGCGAAGAATGCTAAGACCCCTCAGCAGGAG
GAGACAACTTACTACCAAACAGCACTTCCTGGCAATGATAGATACATCAGTGTGGGAAGCCAAGCAGATACAA(SEQ
ID NO：1).
2. the circular rna described in claim 1 is as Down syndrome detection, treatment, the application of prognosis target spot.
3. a kind of Down syndrome detection kit, it is characterised in that test right requirement 1 can be quantified by containing in the kit
The reagent of described circular rna.
4. Down syndrome detection kit according to claim 3, it is characterised in that containing glimmering in real time in the kit
The primer sequence of circular rna described in the quantitative test right requirement 1 of light.
5. Down syndrome detection kit according to claim 4, it is characterised in that the circular rna primer sequence
It is：
F：5’-GCACTTCCTGGCAATGATAGAT-3’(SEQ ID NO：4)；
R：5’-GGCTTGCTGTAGTATCTGGGTG-3’(SEQ ID NO：5).
6. quantitative test right requires application of the reagent of 1 circular rna in Down syndrome detection reagent is prepared.
7. application according to claim 6, it is characterised in that quantitative test right requires that the reagent of 1 circular rna is
The primer sequence of circular rna described in real time fluorescent quantitative test right requirement 1.
8. application according to claim 7, it is characterised in that the circular rna primer sequence is：
F：5’-GCACTTCCTGGCAATGATAGAT-3’(SEQ ID NO：4)；
R：5’-GGCTTGCTGTAGTATCTGGGTG-3’(SEQ ID NO：5).
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