CN102660650A - Reagent kit for measuring absolute value of average length of chromosome telomeres in samples by real-time quantitative PCR (polymerase chain reaction) and reagent kit application - Google Patents
Reagent kit for measuring absolute value of average length of chromosome telomeres in samples by real-time quantitative PCR (polymerase chain reaction) and reagent kit application Download PDFInfo
- Publication number
- CN102660650A CN102660650A CN2012101651617A CN201210165161A CN102660650A CN 102660650 A CN102660650 A CN 102660650A CN 2012101651617 A CN2012101651617 A CN 2012101651617A CN 201210165161 A CN201210165161 A CN 201210165161A CN 102660650 A CN102660650 A CN 102660650A
- Authority
- CN
- China
- Prior art keywords
- telomere
- reverse
- absolute value
- mer
- telomeres
- 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
Images
Landscapes
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention relates to reagent kits, in particular to a reagent kit for measuring absolute value of average length of chromosome telomeres in samples by real-time quantitative PCR (polymerase chain reaction) and a reagent kit application. The reagent kit for measuring absolute value of average length of chromosome telomeres in samples by real-time quantitative PCR (polymerase chain reaction) is characterized by comprising a kit body, a liner, a PCR reaction solution, Taq polymerase, 136B4 Forward, 36B4 Reverse, Telomere Forward, Telomere Reverse, 84-mer and 36B4 fragments. The linear is provided with containers for containing the PCR reaction solution, the Taq polymerase, the 136B4 Forward, the 36B4 Reverse, the Telomere Forward, the Telomere Reverse and the 84-mer respectively. Telomere shortening is supposed to be closely related with cell senescence and diseases such as cancers, telomeres at two ends of each chromosome in cells are shortened at different speed, some telomeres can be shortened greatly suddenly for some reasons, cell senescence and death or diseases such as cancers can be caused when the shortened telomeres exceed a certain limit, and accordingly accurate length measurement to the chromosome telomeres is favorable for preventing cell senescence and cancers. The reagent kit can be used for measuring the absolute value of the average length of the telomeres quickly and conveniently and applicable to industrial production or detection.
Description
Technical field the present invention relates to test kit, the test kit and the application of the absolute value of fringes of chromosome mean length in particularly a kind of real-time quantitative PCR measure sample.
Background technology
Telomere (Telomere) is the section of DNA Tumor-necrosis factor glycoproteins, is positioned at end of chromosome, can keep chromosomal integrity.Nineteen ninety, the length of confirmation telomeres such as Harley can shorten along with increasing of frequency dividing cell gradually.Contact has closely been set up in this discovery between telomere and cell aging.The length of telomere repeat sequence possibly play a part a kind of molecular clock (molecular clock).The people's of different ages the somatic life-span is obviously different, and the length of its telomere is also inequality.Be to shorten with advancing age.Can cultivate for 80-90 generations at subculture in vitro separately from neonatal somatocyte, cultivated for 20～30 generations from 70 years old old man's somatocyte external can only going down to posterity, and the Tumor-necrosis factor glycoproteins length of telomere shorten also a lot.
Telomerase (Telomerase) is to be responsible for a kind of enzyme that telomere prolongs in the cell, is basic nucleoprotein reversed transcriptive enzyme, can telomeric dna be added to the eukaryotic cell end of chromosome.For keeping chromosome stability and cytoactive to play an important role, Telomerase can prolong the telomere (its cellular replication limited ability of the telomere of shortening) that shortens to telomere in the different plant species cell, thereby strengthens the multiplication capacity of cell in vitro.
But in the normal human cell, the activity of Telomerase receives quite tight regulation and control.Experiment showed, the activity that does not have Telomerase in the somatocyte, so the somatocyte per minute splits once, telomere also just shortens.Along with cell constantly divides, the length of telomere is shorter and shorter, and when reaching a critical length, cell dyeing is known from experience loss of stability, cell can not be divided again and gets into apoptosis (apoptosis).The length of telomere has determined the life-span of cell, and therefore the length of the available telomere repeat sequence of losing is inferred the number of times of cell mitogen, so telomere is called as molecular clock or mitotic division clock (mitotic clock).Telomere is except relating to chromosome stability, and is outside the aging and death of cell, also closely related with the generation of tumour.In the intravital various cells of people, the chromosomal end of sexual cell and stem cell grows several thousand base pairs than the end of somatic chromosome, and in these two types of cells, can detect the activity of Telomerase.In addition, all do not record the activity of Telomerase in other all somatocyte.But only exception is to derive from somatic malignant cell to have reappeared telomerase activation; Bring into play the function of its synthetic telomere repeat sequence; Lose to compensate normal telomeric sequence; Make the Tumor-necrosis factor glycoproteins of telomere can not reach the critical length that causes necrocytosis, thereby " immortality " of acquisition cell (immortality).Like this, malignant cell in vivo or external unrestrictedly division growth.
Telomere length mentioned above shortens the mean length that refers to all end of chromosome telomeres.In recent years, a lot of experimental observations begin to query getting in touch between mean length and the aging of telomere.It is by viewpoint single or that certain several short telomere causes that some research groups have proposed in the relevant cell aging of telomere.The exactness of this viewpoint is also proved by increasing experimental data.At present, the research telomere length mainly is to use TRF method (mean length of telomere restriction fragments).In addition, the situation of the short-and-medium telomere of research karyomit(e) that some means can be more accurate is arranged, for example based on the technology of fluorescence in situ hybridization (Q-FISH), perhaps STELA (single telomere length analysis) technology etc.
Summary of the invention
Goal of the invention:
This patent relates to the absolute value test kit and the application thereof of fringes of chromosome mean length in a kind of real-time quantitative PCR measure sample.
A kind of test kit with RT-qPCR method measurement telomere mean length absolute value is characterized in that: comprise box body, liner, PCR reaction solution, Taq enzyme; 136B4 Forward, 36B4 Reverse, Telomere Forward, Telomere Reverse, 84-mer; 36B4 fragment, liner are provided with the container sky and place the PCR reaction solution respectively, Taq enzyme, 136B4 Forward; 36B4 Reverse, Telomere Forward, Telomere Reverse, 84-mer; The nucleotides sequence of wherein said 36B4 Forward is classified as: 5 ' CAG CAA GTG GGA AGG TGT AAT CC 3 ', see Seq NO:1; The nucleotides sequence of 36B4 Reverse is classified as: 5 ' CCC ATT CTA TCA TCA ACG GGT ACA A 3 ', see Seq NO:2; The nucleotides sequence of Telomere Forward is classified as: 5 ' CGG TTT GTT TGG GTT TGG GTT TGG GTT TGG GTT TGG GTT 3 ', see Seq NO:3; The nucleotides sequence of Telomere Reverse is classified as: 5 ' GGC TTG CCT TAC CCT TAC CCT TAC CCT TAC CCT TAC CCT 3 ', see Seq NO:4; The nucleotides sequence of 84-mer is classified as: (TTAGGG)
14Nucleotides sequence classify the nucleotide fragments that contains 14 TTAGGG Tumor-necrosis factor glycoproteinss as, see Seq NO:5; The segmental nucleotides sequence of 36B4 is classified as: 5 ' CAG CAA GTG GGA AGG TGT AAT CCG TCT CCA CAG ACA AGG CCA GGA CTC GTT TGT ACC CGT TGA TGA TAG AAT GGG 3 ', see Seq NO:6.
Described PCR reaction solution contains PCR damping fluid, 1 * SYBR Green master mix, dNTPS.The concentration of primer is 100nM, and the segmental concentration of 84-mer and 36B4 is 1ug/uL;
A kind of application of measuring the test kit of telomere mean length absolute value with the RT-qPCR method is characterized in that realizing set by step:
A. from the human peripheral blood cell, obtain genomic dna;
B. use the described test kit drawing standard of claim 1 curve, chromosomal total copy number in the total length that is used for calculating sample telomere in the RT-qPCR reaction and the reaction: typical curve 1 working concentration gradient is 1,10
-1, 10
-2, 10
-3, 10
-4, 10
-5, 10
-6The C of RT-qPCR reaction of 84-mer oligonucleotide of synthetic
TValue is drawn; Typical curve 2 uses the CT value of artificial synthetic 75 bp 36B4 fragment RT-qPCR reaction to draw, and its concentration gradient is 1,10
-1, 10
-2, 10
-3, 10
-4, 10
-5, 10
-6
C. use telomere fragment and 36B4 gene fragment among the sample gDNA that obtains among the RT-qPCR amplification step A, obtain corresponding C
TValue, and according to the copy number of 36B4 gene in the total length of telomere in the typical curve calculation sample and the sample.
D. telomere mean length absolute value in the calculation sample:
Telomere mean length=telomere total length/36B4 copy number;
Wherein the PCR reaction conditions is:
95 ℃ of sex change 10 min, 95 ℃ of 15sec then, 60 ℃ of 1 min, totally 40 circulations.
Specifically:
1. design of primers: the PCR primer that this patent relates to except routine to be used to the segmental primer of telomere that increases right, also comprise the oligonucleotide fragment (84-mer) of one section 84 bp and the 36B4 fragment of one section 75 bp.
2. drawing standard curve: a) with the 84-mer gradient dilution:: 1,10
-1, 10
-2, 10
-3, 10
-4, 10
-5, 10
-6, 84-mer oligonucleotide chain total length method of calculation (are supposed the undiluted 84-mer 60pg of each reaction needs, promptly 60 * 10 as follows
-12G, the molecular weight of known 84-mer are 26667.2):
1mol=6.02 * 10
23Individual particle number
The quality of an average 84-mer is: 2.6667 * 10
4/ (6.02 * 10
23)=0.44 * 10
-19G;
Undiluted 84-mer total amount is in each PCR reaction system: 60 * 10
-12/ (0.44 * 10
-19)=1.36 * 10
9Individual;
Because the length of 84-mer is 84bp, so undiluted 84-mer total length is in each PCR reaction system: 1.36 * 10
9* 84=1.18 * 10
5Kb;
In like manner can get the total length of different weaker concn 84-mer in each reaction system, drawing standard curve 1.
B) the 36B4 fragment copy number typical curve of drafting 75 bp:
36B4 gradient dilution with 75 bp: 1,10
-1, 10
-2, 10
-3, 10
-4, 10
-5, 10
-6
The 36B4 segment is 75 bp, and relative molecular weight is 23268.1, and single 3,6B4 75 bp fragment quality are 2.32681 * 10
4/ (6.02 * 10
23)=0.38 * 10
-19G;
The 36B4 content of maximum concentration 75 bp is 200 pg in the assumed response system, and the copy number of 75 bp 36B4 is in each reaction system so: 200 * 10
-12/ 0.38 * 10
-19=5.26 * 10
9Be equivalent in diplontic genome, contain 5.26 * 10
9/ 2=2.63 * 10
9Individual 36B4 gene copy.
In like manner can get 75 bp 36B4 copy numbers in the different weaker concn reaction systems, make typical curve 2.
3. telomere and 36B4 gene in the RT-qPCR amplification gDNA sample;
4. telomere and 36B4 gene (confidential reference items) in the RT-QPCR amplification gDNA sample, and the total copy number of gDNA in total length through telomere in two typical curves calculating gDNA samples and the sample, promptly can calculate the telomere mean length:
The total copy number of telomere total length/gDNA in telomere mean length=sample
The principle explanation:
1, real-time fluorescence quantitative PCR (RT-qPCR) principle
No matter be that inherited disease (like thalassemia and hemophilia), transmissible disease (like hepatitis and AIDS) or tumour are carried out gene diagnosis; Still study the influence of medicine to gene expression dose; Perhaps monitor the result of treatment of medicine and therapy, quantitative PCR technique can be brought into play great role.The latest developments of quantitative PCR technique are real time fluorescent quantitatives.This technology detects the PCR product by means of fluorescent signal; Improved sensitivity on the one hand; Can also accomplish that on the other hand the every circulation primary of PCR just collects data, set up real-time amplification curve, confirm the CT value exactly; Thereby confirm the initiate dna copy number according to the CT value, accomplished that DNA truly is quantitative.Different according to the data that finally obtain, quantitative PCR can be divided into two kinds of relative quantification and absolute quantitation.Through the height variation of gene expression dose in two samples of different modes processing, the result who obtains is a per-cent to typical relative quantification as relatively; Absolute quantitation then need use typical curve to confirm the copy number or the concentration of gene in the sample.
The definition of CT value is: when the fluorescent signal of amplified production reaches preset threshold the amplification cycles number of process.
The mathematical principle of quantitative PCR
Ideal PCR reaction:
X=X
0×2
n
Nonideal PCR reaction:
X=X
0（1+Et）
n；
N: the cycle index of amplified reaction
X: the product amount after the n time circulation
X
0: the original template amount
Ex: amplification efficiency
When amplified production reaches threshold line:
X
CT=X
0(1+Ex)
CT, （1）
X wherein
CT: be the amount of amplified fluorescence signal time that reaches threshold intensity institute's amplified production, after threshold line was set, it was a constant, is set at M;
Take the logarithm simultaneously in equation (1) both sides:
logM=logX
0(1+Ex)
CT （2）
Arrangement equation (2):
logX
0=-log(1+Ex)*CT+logM (3)
LogX
0Linear with CT, the CT value of amplification can calculate the contained template amount in the sample per sample.
2, SYBR Green I optical dye know-why SYBR Green I is a kind of the optical dye with the dna double chain combination.When it and dna double chain combination, send fluorescence; When the dna double chain discharged, fluorescent signal sharply weakened.Therefore, in an individual system, its strength of signal has been represented the quantity of double chain DNA molecule.The primary process of SYBR Green optical dye standard measure PCR is: 1, begin reaction, when SYBR Green dyestuff and dna double chain combination, send fluorescence.2, during the DNA sex change, SYBR Green dyestuff discharges, and fluorescence sharply reduces.3, in polymerization extension process, primer annealing also forms the PCR product.4, after polymerization was accomplished, SYBR Green dyestuff combined with double-stranded product, and the amount of having a net increase of that the quantitative PCR system detects fluorescence strengthens.
2, RT-qPCR measures the telomere length principle
As shown in Figure 5, green area is the telomere district, i.e. (TTAGGG) n Tumor-necrosis factor glycoproteins; Blue arrow is and telomere specificity bonded primer.Because be proportionate with the length of telomere bonded primer quantity heel end grain, telomere is long, its combinative primer quantity is many, and fluorescence intensity is big, be appreciated that into copy number many; Otherwise then copy number is few, a little less than the fluorescence intensity.So use the RT-qPCR method, obtain the CT value of each sample, can reflect the mean length of telomere in each sample indirectly.
Beneficial effect:
The shortening of telomere is considered to the generation of diseases such as the aging of cell and cancer closely related.Simultaneously, the telomere at every karyomit(e) two ends is not to shorten with identical speed in the cell, and the telomere that has forms short relatively telomere because some reason can shorten suddenly significantly.When these short telomeres meet or exceed a certain limit, will cause cell decline dead, perhaps like the disease of cancer etc.Therefore, detect the cell telomere length exactly, can both play very large help the generation that prevents old and feeble and preventing cancer.The present invention adopts kit form to make things convenient for, and fast measures the absolute value of the mean length of telomere, is suitable for industrial production or detection.
Description of drawings:
Fig. 1. typical curve 1, be used for calculating the total length that each RT-qPCR reacts telomere, transverse axis is a telomere length, unit is Kb, the C of PCR reaction
TValue is linear in scope shown in Figure 1 with telomere length.Through optimizing the concentration of sample, the CT value of its PCR reaction is dropped in the typical curve linearity range, should this use this typical curve calculating institute value to equal the total length of telomere in each PCR reaction.
Fig. 2. typical curve 2 is used for calculating the copy number of 36B4 in each reaction, chromosomal copy number in each reaction of indirect calculation, i.e. total copy number of telomere in each PCR reaction.Transverse axis is the copy number of 36B4, and the CT value of PCR reaction and the LOG value of 36B4 copy number are linear in the scope shown on the way.
Fig. 3. the dependency of the telomere mean length absolute value that use RT-qPCR method and TRF method record.
Fig. 4 is a synoptic diagram of the present invention, and wherein box body 1, liner 2, and PCR reaction solution 3, Taq enzyme 4,136B4 Forward 5,36B4 Reverse 6, Telomere Forward 7, Telomere Revers e 9,84-mer 10,36B4 fragment 1.
Fig. 5. the RT-qPCR method detects the synoptic diagram of telomere length, and the zone that circle is wherein arranged is for blue, and no encircled is green.
Embodiment
RT-QPCR uses the PCR reaction solution available from Qiagen.
The extraction of embodiment 1 peripheral blood genomic dna (gDNA)
1). blood sample is distributed into 1 mL/ pipe, in-70 ℃ of storages.
2). sample is thawed, and every pipe sample adds 0.8 mL citrate buffer solution, and 12000 leave the heart 1 min behind the mixing, remove supernatant.
3). add 1 mL, 1 * citrate buffer solution, concussion back 12000 leaves the heart 1 min, removes supernatant.
4). add the Na of 375 uL 0.2M
2OAc, the concussion back adds SDS and 5 uL proteinase K (the 20 mg/mL H of 25 uL 10%
2O), 55 degree are hatched 1 h behind the concussion mixing.
5). add 120 uL phenol/chloroform/primary isoamyl alcohol (v/v:25/24/1), leave the heart 2 min in 12000 after shaking 30 sec.
6). it is secondary that aqueous phase layer is moved to new 1.5 mL centrifuge tubes, adds 1 mL, 100% cold ethanol, mixing is also hatched 15 min in-20 degree.
7). 12000 leave the heart 2 min, go supernatant and oven dry.
8). add the TE damping fluid of 180 uL 10:1,10 min are hatched in 55 ℃ in the concussion back.
9). add 20 uL, the sodium acetate soln of 2M adds 500 uL behind the mixing, and 100% cold ethanol leaves the heart 1 min in 12000 behind the mixing, removes supernatant.
10). use 1 mL, 80% washing with alcohol deposition leaves the heart 1 min in 12000 afterwards, removes supernatant.11). vacuum-drying precipitates 10 min.
12). will precipitate and be dissolved in again in the 200 uL TE damping fluids, 55 ℃ of incubated overnight obtain gDNA.
Embodiment 2The RT-QPCR method is surveyed the absolute value of telomere mean length
1, a kind of test kit with RT-qPCR method measurement telomere mean length absolute value comprises box body 1, liner 2, PCR reaction solution 3; Taq enzyme 4,136B4 Forward 5,36B4 Reverse 6, Telomere Forward 7; Telomere Revers e 9,84-mer 10,36B4 fragment 11, liner 2 is provided with container cavity and places PCR reaction solution 3 respectively; Taq enzyme 4,136B4 Forward 5,36B4 Reverse 6, Telomere Forward 7; Telomere Reverse 8,84-mer 10,36B4 fragment 11;
The nucleotides sequence of wherein said 36B4 Forward is classified as: 5 ' CAG CAA GTG GGA AGG TGT AAT CC 3 ';
The nucleotides sequence of 36B4 Reverse is classified as: 5 ' CCC ATT CTA TCA TCA ACG GGT ACA A 3 ';
The nucleotides sequence of Telomere Forward is classified as: 5 ' CGG TTT GTT TGG GTT TGG GTT TGG GTT TGG GTT TGG GTT 3 ';
The nucleotides sequence of Telomere Reverse is classified as: 5 ' GGC TTG CCT TAC CCT TAC CCT TAC CCT TAC CCT TAC CCT 3 ';
The nucleotides sequence of 84-mer is classified as: (TTAGGG)
14Nucleotides sequence classify the nucleotide fragments that contains 14 TTAGGG Tumor-necrosis factor glycoproteinss as;
The segmental nucleotides sequence of 36B4 is classified as: 5 ' CAG CAA GTG GGA AGG TGT AAT CCG TCT CCA CAG ACA AGG CCA GGA CTC GTT TGT ACC CGT TGA TGA TAG AAT GGG 3 '.
2, utilize 84-mer to draw the total length typical curve
1), the RT-qPCR reaction conditions of 84-mer: the RT-qPCR reaction system is 20 uL; With 84-mer is template, gets 84-mer 1 uL, 100 nM primer Telomere Forward, 1 uL; 100 nM primer Telomere Reverse1uL, surplus is the PCR reaction solution.RT-qPCR reaction conditions: 95 ℃ of 10 min, 40 circulations (95 ℃ of 15 sec, 60 ℃ of 1 min)
2), drawing standard curve
A) with the 84-mer gradient dilution: 1,10
-1, 10
-2, 10
-3, 10
-4, 10
-5, 10
-6
84-mer oligonucleotide chain total length method of calculation are following:
Suppose the undiluted 84-mer 60pg of each reaction needs, promptly 60 * 10
-12G, the relative molecular weight of known 84-mer are 26667.2;
The quality of an average 84-mer is: 2.6667 * 10
4/ 6.02 * 10
23=0.44 * 10
-19
Undiluted 84-mer total amount is in each PCR reaction system: 60 * 10
-12/ 0.44 * 10
-19=1.36 * 10
9, so undiluted 84-mer total length is in each PCR reaction system: 1.36 * 10
9* 84=1.18 * 10
5Kb;
In like manner can get the total length of different weaker concn 84-mer in each reaction system, drawing standard curve 1 (Fig. 1), the typical curve equation is: log (TL)=-4.86C
T+ 40.14, wherein TL is a total length.
3, utilize 36B4 drawing standard curve
1) the RT-qPCR reaction conditions of 3,6B4 75 bp: the RT-qPCR reaction system is 20 uL, is template with 36B4, gets 36B4 1uL, 100 nM 36B4 Forward 1uL, and 100 nM 36B4 Reverse 1uL, surplus is the PCR reaction solution.RT-qPCR reaction conditions: 95 ℃ of 10 min, 40 circulations (95 ℃ of 15 sec, 60 ℃ of 1 min)
36B4 gradient dilution with 75 bp: 1,10
-1, 10
-2, 10
-3, 10
-4, 10
-5, 10
-6
36B4 is 75 bp, and relative molecular weight is 23268.1;
An average 3,6B4 75 bp quality are 2.32681 * 10
4/ 6.02 * 10
23=0.38 * 10
-19G;
Maximum concentration 75 bp 36B4 content are 200 pg in the assumed response system, and the copy number of 75 bp 36B4 is in each reaction system so: 200 * 10
-12/ 0.38 * 10
-19/ 2=2.63 * 10
9
In like manner can get 75 bp 36B4 copy numbers in the different weaker concn reaction systems, make typical curve 2 (Fig. 2).
The typical curve equation is: log (TC)=-5.11C
T+ 39.57, wherein TC is the copy number of 36B4 gene.Because the 36B4 gene is a single copy gene, be to do one of RT-qPCR internal control gene commonly used, so known that the copy number of 36B4 just equals to have known chromosomal copy number in the sample among the gDNA, just can calculate the absolute value of telomere mean length.
4, the 36B4 (confidential reference items) of telomere and 75 bp in the RT-qPCR amplification gDNA sample
The RT-qPCR reaction conditions of embodiment 1 telomere in the gDNA sample that obtains: the RT-qPCR reaction system is 20 uL; GDNA 1 uL; 100 nM Telomere Forward, 1 uL; 100 nM Telomere Reverse, 1 uL (the primer here is the same with the primer of 84mer), surplus is the PCR reaction solution; The PCR reaction conditions is: 95 ℃ of 10 min, 40 circulations (95 ℃ of 15 sec, 60 ℃ of 1 min).
The RT-qPCR reaction conditions of the 36B4 of 75 bp in the step g DNA sample: the RT-qPCR reaction system is 20 uL; 3,6B4 1 uL wherein; 100 nM primer 36B4 Forward, 1 uL, 100 nM primer 36B4 Reverse, 1 uL, surplus is the PCR reaction solution.RT-qPCR reaction conditions: 95 ℃ of 10 min, 40 circulations (95 ℃ of 15 sec, 60 ℃ of 1 min).
C with telomere and 36B4 in the gDNA sample of step (4) acquisition
TValue, two typical curve equations of substitution, i.e. log (TL)=(C respectively
T (telomere)-40.14)/-4.86, wherein TL is a telomere total length in the sample; LOG (TC)=(C
T (36B4)-39.57)/-5.11, wherein TC is a telomere copy number in the sample, promptly can calculate telomere mean length=telomere total length/telomere copy number.
Because 84mer is (TTAGGG)
14, be equivalent to the telomere repeat sequence of synthetic, with telomeric sequence and the indistinction in the cell, be the length that can be used for the real telomere of calculation sample so do typical curve with artificial synthetic telomeric sequence; In like manner the 36B4 fragment of synthetic also can be used for the 36B4 copy number in the calculation sample.In addition; The formula that calculates mean length is that a simple mathematical is calculated; Such as per day mileage=total actual service life/fate of a running car, such formula need not be derived in detail, in like manner; Relation between the copy number of the total length of the mean length of telomere, telomere and telomere in the sample (being the chromosome copies number) also is conspicuous, need not to derive.
Comparative Examples: the TRF method survey telomere mean length (with reference to Aubert etc., Telomere length measurement-Caveats and a critical assessment of the available technologies and tools. Mutat Res. 2012 Feb 1; 730 (1-2): 59-67.)
1). with 0.5 ug gDNA sample with the complete digestion of Hae III (available from NEB) enzyme;
2). postdigestive gDNA fragment is separated by agarose gel electrophoresis, is transferred to then on the nylon membrane;
3). use radioactivity
32The probe of P mark
32P-(TTAGGG)
7With the telomere fragment hybridization on the nylon membrane;
4). detect the segmental length of each sample telomere through radioautograph.
The telomere length that the TRF method records is taken the logarithm; The sample telomere mean length absolute value that then itself and RT-qPCR method is recorded is figure; Can get as shown in Figure 3; The Trendline of each data point is a straight line, shows that the measured telomere mean length absolute value of RT-qPCR method that this patent relates to has very strong dependency with the classical measured result of TRF method, is reliable result.
Claims (3)
1. the test kit with RT-qPCR method measurement telomere mean length absolute value is characterized in that: comprise box body, liner, PCR reaction solution, Taq enzyme; 136B4 Forward, 36B4 Reverse, Telomere Forward, Telomere Reverse, 84-mer; 36B4 fragment, liner are provided with the container sky and place the PCR reaction solution respectively, Taq enzyme, 136B4 Forward; 36B4 Reverse, Telomere Forward, Telomere Reverse, 84-mer;
The nucleotides sequence of wherein said 36B4 Forward is classified Seq NO:1 as;
The nucleotides sequence of 36B4 Reverse is classified Seq NO:2 as;
The nucleotides sequence of Telomere Forward is classified as and is seen Seq NO:3;
The nucleotides sequence of Telomere Reverse is classified Seq NO:4 as;
The nucleotides sequence of 84-mer is classified Seq NO:5 as;
The segmental nucleotides sequence of 36B4 is classified as: see Seq NO:6.
2. a kind of test kit with RT-qPCR method measurement telomere mean length absolute value according to claim 1, it is characterized in that: described PCR reaction solution contains PCR damping fluid, 1 * SYBR Green master mix, dNTPs.
3. application of measuring the test kit of telomere mean length absolute value with the RT-qPCR method is characterized in that realizing set by step:
From the human peripheral blood cell, obtain genomic dna;
Use the described test kit drawing standard of claim 1 curve, chromosomal total copy number in the total length that is used for calculating sample telomere in the RT-qPCR reaction and the reaction: typical curve 1 working concentration gradient is 1,10
-1, 10
-2, 10
-3, 10
-4, 10
-5, 10
-6The 84-mer oligonucleotide of synthetic as template, the C of RT-qPCR reaction
TValue is drawn; Typical curve 2 uses artificial synthetic 36B4 fragment as template, and the CT value of RT-qPCR reaction is drawn, and its concentration gradient is 1,10
-1, 10
-2, 10
-3, 10
-4, 10
-5, 10
-6Use telomere fragment and 36B4 gene fragment among the sample gDNA that obtains among the RT-qPCR amplification step A, obtain corresponding C
TValue, and according to the copy number of 36B4 gene in the total length of telomere in the typical curve calculation sample and the sample; Telomere mean length absolute value in the calculation sample: telomere mean length=telomere total length/36B4 copy number; Wherein the PCR reaction conditions is: 95 ℃ of sex change 10 min, 95 ℃ of 15sec then, 60 ℃ of 1 min, totally 40 circulations.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2012101651617A CN102660650A (en) | 2012-05-25 | 2012-05-25 | Reagent kit for measuring absolute value of average length of chromosome telomeres in samples by real-time quantitative PCR (polymerase chain reaction) and reagent kit application |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2012101651617A CN102660650A (en) | 2012-05-25 | 2012-05-25 | Reagent kit for measuring absolute value of average length of chromosome telomeres in samples by real-time quantitative PCR (polymerase chain reaction) and reagent kit application |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN102660650A true CN102660650A (en) | 2012-09-12 |
Family
ID=46770225
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN2012101651617A Pending CN102660650A (en) | 2012-05-25 | 2012-05-25 | Reagent kit for measuring absolute value of average length of chromosome telomeres in samples by real-time quantitative PCR (polymerase chain reaction) and reagent kit application |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN102660650A (en) |
Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103233071A (en) * | 2013-04-27 | 2013-08-07 | 南京优而生物科技发展有限公司 | Method for measuring telomere absolute length |
| CN106755429A (en) * | 2016-12-27 | 2017-05-31 | 上海三誉生物科技有限公司 | A kind of method of accurate measurement crowd telomere length |
| CN108342474A (en) * | 2018-04-13 | 2018-07-31 | 东华大学 | A kind of kit for detecting telomere length prediction coronary heart disease risk |
| CN109943626A (en) * | 2019-04-18 | 2019-06-28 | 潍坊峡山荆卫生物科技有限公司 | A kind of PCR method detecting absolute telomere length |
| CN113557307A (en) * | 2018-12-26 | 2021-10-26 | 生命长度有限公司 | Method for determining telomere-related variables and use thereof for diagnosis and/or prognosis of telomere-related diseases |
| CN116555402A (en) * | 2023-06-27 | 2023-08-08 | 成都云测医学生物技术有限公司 | Telomere detection kit and telomere length detection method for non-disease diagnosis purpose |
| CN118272504A (en) * | 2024-05-31 | 2024-07-02 | 赛奥斯博生物科技(北京)有限公司 | Primer, probe, method and application for detecting vector copy number based on reference gene |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1427895A (en) * | 2000-03-07 | 2003-07-02 | 英国国防部 | Method for analysing length of nucleic acid molecule |
| CN101193643A (en) * | 2005-03-25 | 2008-06-04 | Alt解决方案公司 | Modulation of telomere length in telomerase positive cells and cancer therapy |
| WO2008119027A2 (en) * | 2007-03-27 | 2008-10-02 | The Johns Hopkins University | Genetic testing of telomere-associated genes in dyskeratosis congenita and related disorders |
| WO2011086161A2 (en) * | 2010-01-15 | 2011-07-21 | Facultes Universitaires Notre-Dame De La Paix | Biomarkers of ageing |
| CN102296113A (en) * | 2011-08-12 | 2011-12-28 | 杨楠 | Method for measuring absolute length of chromosome telomere by monochrome multiplex quantitative PCR method |
-
2012
- 2012-05-25 CN CN2012101651617A patent/CN102660650A/en active Pending
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1427895A (en) * | 2000-03-07 | 2003-07-02 | 英国国防部 | Method for analysing length of nucleic acid molecule |
| CN101193643A (en) * | 2005-03-25 | 2008-06-04 | Alt解决方案公司 | Modulation of telomere length in telomerase positive cells and cancer therapy |
| WO2008119027A2 (en) * | 2007-03-27 | 2008-10-02 | The Johns Hopkins University | Genetic testing of telomere-associated genes in dyskeratosis congenita and related disorders |
| WO2011086161A2 (en) * | 2010-01-15 | 2011-07-21 | Facultes Universitaires Notre-Dame De La Paix | Biomarkers of ageing |
| CN102296113A (en) * | 2011-08-12 | 2011-12-28 | 杨楠 | Method for measuring absolute length of chromosome telomere by monochrome multiplex quantitative PCR method |
Non-Patent Citations (3)
| Title |
|---|
| NATHAN J O’CALLAGHAN, M.FENECH: "A quantitative PCR method for measuring", 《BIOLOGICAL PROCEDURES ONLINE》, vol. 13, no. 3, 31 January 2011 (2011-01-31) * |
| 郭令军，张七一: "实时定量PCR分析外周血白细胞的端粒长度", 《中外医疗》, no. 21, 31 December 2008 (2008-12-31) * |
| 钟英成 等: "荧光实时定量PCR检测端粒长度", 《临床和实验医学杂志》, vol. 7, no. 4, 30 April 2008 (2008-04-30) * |
Cited By (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103233071A (en) * | 2013-04-27 | 2013-08-07 | 南京优而生物科技发展有限公司 | Method for measuring telomere absolute length |
| CN106755429A (en) * | 2016-12-27 | 2017-05-31 | 上海三誉生物科技有限公司 | A kind of method of accurate measurement crowd telomere length |
| CN108342474A (en) * | 2018-04-13 | 2018-07-31 | 东华大学 | A kind of kit for detecting telomere length prediction coronary heart disease risk |
| CN113557307A (en) * | 2018-12-26 | 2021-10-26 | 生命长度有限公司 | Method for determining telomere-related variables and use thereof for diagnosis and/or prognosis of telomere-related diseases |
| CN113557307B (en) * | 2018-12-26 | 2024-05-28 | 生命长度有限公司 | Method for determining telomere-related variable and use thereof in diagnosis and/or prognosis of telomere-related diseases |
| CN109943626A (en) * | 2019-04-18 | 2019-06-28 | 潍坊峡山荆卫生物科技有限公司 | A kind of PCR method detecting absolute telomere length |
| CN116555402A (en) * | 2023-06-27 | 2023-08-08 | 成都云测医学生物技术有限公司 | Telomere detection kit and telomere length detection method for non-disease diagnosis purpose |
| CN116555402B (en) * | 2023-06-27 | 2023-09-26 | 成都云测医学生物技术有限公司 | Telomere detection kit and telomere length detection method for non-disease diagnosis purpose |
| CN118272504A (en) * | 2024-05-31 | 2024-07-02 | 赛奥斯博生物科技(北京)有限公司 | Primer, probe, method and application for detecting vector copy number based on reference gene |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN102660650A (en) | Reagent kit for measuring absolute value of average length of chromosome telomeres in samples by real-time quantitative PCR (polymerase chain reaction) and reagent kit application | |
| CA2513747C (en) | Methods of predicting mortality risk by determining telomere length | |
| ES2762860T3 (en) | Measures of abundance of short telomeres | |
| Samsonraj et al. | Telomere length analysis of human mesenchymal stem cells by quantitative PCR | |
| JP7175326B2 (en) | Target nucleic acid amplification method and target nucleic acid amplification composition | |
| WO2009133915A1 (en) | Cancer marker, method for evaluation of cancer by using the cancer marker, and evaluation reagent | |
| WO2016186987A1 (en) | Biomarker micrornas and method for determining tumor burden | |
| CN106480021A (en) | A kind of nucleic acid constant-temperature exponential amplification techniques based on symmetric annular dumbbell template and its application in microRNA detection | |
| CN104328164A (en) | Kit for detecting human EGFR gene mutation by using fluorescence probe hybridization method | |
| CN202558870U (en) | Kit for quantifying absolute value of mean length of chromosome telomere in PCR (Polymerase Chain Reaction) measurement sample in real time | |
| CN110616229A (en) | Fusion gene of FGFR1 translocation blood disease and detection primer and application thereof | |
| CN108004299B (en) | Fluorescent quantitative in situ hybridization (Q-FISH) method for determining telomere length by using genome DNA | |
| Gentilini et al. | Retrospective monitoring of minimal residual disease using hairpin-shaped clone specific primers in B-cell lymphoma affected dogs | |
| ES2691306T3 (en) | Procedures and compositions for detecting mutations in the human PI3KCA (PIK3CA) gene | |
| CA2325343A1 (en) | Quantitative analysis of gene expression | |
| JP2001204483A (en) | DETERMINATION OF hTERT mRNA EXPRESSION | |
| ES2656961T3 (en) | Use of G-Clamp to improve allele-specific PCR | |
| ES2536195T3 (en) | Sensitivity to angiogenesis inhibitors | |
| CN102618633A (en) | Method for detecting length of the shortest telomere in cells by using improved STELA method | |
| CA2984516C (en) | Method, sequences, compositions and kit for detection of changes in the promoter of the gene htert | |
| JPWO2018101375A1 (en) | Human genomic DNA detection method | |
| CN107058591A (en) | Telomere length detection method and kit | |
| HUE027213T2 (en) | Method for identification of sensitivity of a patient to telomerase inhibition therapy | |
| Peters et al. | Quantitative polymerase chain reaction-based assay with fluorogenic Y-chromosome specific probes to measure bone marrow chimerism in mice | |
| CN101509040B (en) | Reagent kit for inosculating status analysis after hemopoietic stem cell transplantation and uses thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C02 | Deemed withdrawal of patent application after publication (patent law 2001) | ||
| WD01 | Invention patent application deemed withdrawn after publication |
Application publication date: 20120912 |