CN105256038A - PCR detecting primer and kit for genes of heterocephalus-glaber cell factor IL-6 - Google Patents

Abstract

The invention relates to a technology for detecting genes of a heterocephalus-glaber cell factor IL-6, belongs to the field of biotechnology detection, and provides a PCR detecting primer and kit for genes of the heterocephalus-glaber cell factor IL-6. The forward primer is 5'-ACGCTAGTCCTCCACGAT-3', and the reverse primer is 5'-GGTTGTTTAACATTGCCTTT-3'. The invention further provides a method for detecting the genes of the heterocephalus-glaber cell factor IL-6. The method includes the steps that total mRNA of macrophage of heterocephalus glaber is extracted and subjected to inverse transcription to form cDNA which serves as a detected sample, detection is carried out with a Real-time PCR method, the expression level of the IL-6 is obtained, and the expression of the genes of the IL-6 is quantitatively and qualitatively detected with a PCR detection technology. The primer and the method are simple, rapid and suitable for use and popularization.

Description

Naked mole cytokine IL-6 gene PCR detects primer and detection kit

Technical field

The invention belongs to field of biological technology detection, be specifically related to naked mole cytokine IL-6 technique of gene detection.

Background technology

Naked mole (Heterocephalusglaber, nakedmolerat, NMR) be a kind of special rodent, its life-span is the longest in rodent, the oldest individuality is more than 3O year, 2O year about animal bodies state generally good, the sign that the naked mole performance more than 25 years old is weak, mostly dies from senile disease; The situation that naked mole dies from cancer never finds, anatomical results also never finds tumour, has certain anticancer mechanism.The exploration of naked mole antitumor properties mechanism opens new chapter by giving the research of tumour.At present, a lot of bases, clinical and EPDML research are verified, and inflammation is one clearly can lead oncogenic Hazard Factor.The mechanism that etiology causes certain specific tumors to be fallen ill also is not suitable for other cancer kinds, but pathogenesis is in essence all identical, is exactly induction and the maintenance of inflammation.Interleukin 6 (interleukin-6, IL-6) (the GeneID:3569 of people IL-6, the GeneID:16193 of mouse IL-6) can break up and produce antibody by elicit B cell, and inducing T cell activation is bred, differentiation, participate in the immunne response of body, be Inflammatory response inspire agent.Therefore, the mechanism disclosing the antitumor properties that it may exist is conducive to the detection of naked mole IL-6 gene expression dose.

At present, due to naked mole sexual maturity, (Female sexual maturation age is 228 days, is 6.5 times of mouse evening; Male sexual maturation age is 12 months, is 8.1 times of mouse), the Gestation period long (68 days is 3.2 times of mouse), and wild naked mole lives in sweltering heat, area, dry East Africa, throughout one's life in the underground of dark, is difficult to catch.Therefore, the number of naked mole is relatively less, greatly constrains its applied research.The cell of vitro culture has division and multiplication capacity, strong adaptability, easily the feature such as cultivation, and therefore, somatic cell culture technology can solve naked mole research material and to originate less problem.

At present, in research, the detection of cytokine IL-6 genetic expression is carried out mainly through ELISA detection kit, in the method, need using specific antibody, but at present still not for the specific antibody of naked mole.

Summary of the invention

In order to solve above-mentioned the deficiencies in the prior art, the invention provides primer and the detection kit of naked mole cytokine IL-6 gene PCR detection, utilize PCR detection technique quantitatively and qualitatively to detect IL-6 genetic expression, simple and fast, is applicable to promoting the use of.

A first aspect of the present invention, provides a kind of naked mole cytokine IL-6 gene PCR and detects primer:

Forward primer: 5 '-ACGCTAGTCCTCCACGAT-3 ' (SEQIDNO:1),

Reverse primer: 5 '-GGTTGTTTAACATTGCCTTT-3 ' (SEQIDNO:2).

A second aspect of the present invention, provides a kind of naked mole cytokine IL-6 gene PCR detection kit, and its detection primer is:

Forward primer: as shown in SEQIDNO:1,

Reverse primer: as shown in SEQIDNO:2.

Above-mentioned naked mole cytokine IL-6 gene PCR detection kit, also comprises:

2×SYBRPremixExTaq5ul，ROXReferenceDye(50×)0.2ul，ddH ₂O3.6ul。

Preferably, one of the present invention naked mole cytokine IL-6 gene PCR detection kit, comprising:

Forward primer: as shown in SEQIDNO:1,

Reverse primer: as shown in SEQIDNO:2;

2×SYBRPremixExTaq5ul；

ROXReferenceDye(50×)0.2ul；

ddH ₂O3.6ul。

A third aspect of the present invention, provide a kind of detection method of naked mole cytokine IL-6 gene, described detection method utilizes above-mentioned specific detection primer; Or utilizing above-mentioned detection kit, described detection method comprises the steps:

Extract total mRNA of naked mole Macrophage Cell, and reverse transcription is cDNA as detection sample, use Real-timePCR method to detect, draw the expression level of IL-6, primer sequence is as follows:

Forward primer: as shown in SEQIDNO:1,

Reverse primer: as shown in SEQIDNO:2;

The detection method of above-mentioned naked mole cytokine IL-6 gene, concrete detecting step is:

1) mRNA is extracted:

The naked mole scavenger cell of separation and Culture, extract cell total rna, and reverse transcription is cDNA.

2) real-time fluorescence quantitative PCR reaction system 10ul is prepared

Get step 1) middle cDNA template 0.8ul, 2 × SYBRPremixExTaq5ul, PCR forward primer (10 μ Μ) 0.2ul, PCR reverse primer (10uM) 0.2ul, ROXReferenceDye (50 ×) 0.2ul, ddH ₂o3.6ul.Negative control is set.

3) PCR reaction is carried out

Reaction cycle is set to: 95 DEG C of 30s cycles stage of holding stage, 95 DEG C of 5s, 60 DEG C of 34s, 72 DEG C of 30s, 40 circulations; Solubility curve stage 95 DEG C of 15s, 60 DEG C of 1min, 95 DEG C of 15s, 40 circulations.

Technique effect of the present invention is as follows:

Primer used detects for the mRNA level in-site of naked mole IL-6, extract total mRNA of naked mole Macrophage Cell, and reverse transcription is that cDNA is as detection sample, Real-timePCR method is used to detect, draw the expression level of IL-6, as judging the basis for estimation that naked mole IL-6 genetic expression changes, there is good specificity and practicality.

Accompanying drawing illustrates:

Fig. 1 is the melting curve that embodiment 1 real-time fluorescence quantitative PCR detects;

Fig. 2 is the amplification curve that embodiment 1 real-time fluorescence quantitative PCR detects;

Fig. 3 is (b, c, melting curves d) detected with primer sequence (a) real-time fluorescence quantitative PCR in the present invention of other three pairs of primers in embodiment 2.

Embodiment

Concrete the present invention is further illustrated with accompanying drawing in conjunction with the embodiments, but enforcement of the present invention is not limited in this.

Embodiment 1:

MRNA sequence according to mole cytokine IL-6 gene naked on GeneBank carries out design of primers, and primer sequence is as follows:

Forward primer: 5 '-ACGCTAGTCCTCCACGAT-3 ' (SEQIDNO:1)

Reverse primer: 5 '-GGTTGTTTAACATTGCCTTT-3 ' (SEQIDNO:2).

Concrete detecting step is as follows:

1) mRNA is extracted:

The naked mole scavenger cell of separation and Culture, A group is blank group, is labeled as 1,2; B group is experimental group, uses virus mimics polyI:C stimulating expression of macrophage, is labeled as 3-6.Extract cell total rna (TaKaRaRNAisoPlus) (TangXL respectively, XinY, etal.MetabolicCharacteristicsandResponsetoHighAltitudein Phrynocephaluserythrurus (Lacertilia:Agamidae), aLizardDwellatAltitudesHigherThanAnyOtherLivingLizardsin theWorld.PLoSOne.2013Aug7; , and reverse transcription is cDNA (FastQuantRTKit (WithgDNase) (KR106), purchased from Tian Gen biochemical technology company limited) 8 (8): e71976.).

2) real-time fluorescence quantitative PCR reaction system 10ul is prepared

Get step 1) middle cDNA template 0.8ul, 2 × SYBRPremixExTaq5ul, PCR forward primer (PCRForwardprimer) (10 μ Μ) 0.2ul, PCR reverse primer (PCRreverseprimer) (10uM) 0.2ul, ROXReferenceDye (50 ×) 0.2ul, redistilled water (ddH ₂o) 3.6ul.Negative control is set.

3) PCR reaction is carried out

Reaction cycle is set to: holding stage (Holdingstage) 95 DEG C of 30s; Cycle stage (cyclingstage) 95 DEG C of 5s, 60 DEG C of 34s, 72 DEG C of 30s, 40 circulations; Solubility curve stage (Meltcurvestage) 95 DEG C of 15s, 60 DEG C of 1min, 95 DEG C of 15s, 40 circulations.

Fig. 1 melting curve represents the specificity (in figure, curve 1,2 represents blank group, and curve 3,4,5,6 represents polyI:C stimulating group) of IL-6 factor primer amplification; Fig. 2 amplification curve represents the real-time fluorescence quantitative PCR amplification curve of blank group (1,2) and polyI:C stimulating group (3,4,5,6).

In the melting curve figure of Fig. 1 embodiment 1, all there is single peak value in all sample PCR reactions of detection, solvent temperature Tm value is 85.15 DEG C, illustrates that quantitative fluorescent PCR reacts IL-6 primer used and has good specificity.In Fig. 2 amplification curve, ordinate zou Δ Rn refers to standard indicator signal value and deducts baseline signal value, and X-coordinate represents cycle number, as seen from the figure, what the Δ Rn value of all samples detected all counted up at PCR circulating ring advances into plateau, illustrates that real-time fluorescence quantitative PCR reaction result is effective.

Embodiment 2

MRNA sequence information according to mole cytokine IL-6 gene naked on GeneBank designs other three kinds of primers simultaneously:

b：

Forward primer 5 '-CGTCAAGTAAGCCAACAA-3 ' (SEQIDNO:3),

Reverse primer 3 '-GGAGGACTAGCGTAAACAG-5 ' (SEQIDNO:4).

c：

Forward primer: 5 '-TTACGCTAGTCCTCCACG-3 ' (SEQIDNO:5),

Reverse primer: 3 '-GGTTGTTTAACATTGCCTTT-5 ' (SEQIDNO:2).

d：

Forward primer: 5 '-ATTTCAGGGCTGATACGA-3 ' (SEQIDNO:6),

Reverse primer: 3 '-TTCAGTTCCTGGACATCG-5 ' (SEQIDNO:7).

Concrete detecting step is as follows:

1) mRNA is extracted:

The naked mole scavenger cell of separation and Culture, extract cell total rna (TaKaRaRNAisoPlus), and reverse transcription is cDNA (FastQuantRTKit (WithgDNase) (KR106), purchased from Tian Gen biochemical technology company limited).

2) real-time fluorescence quantitative PCR reaction system 10ul is prepared

Get step 1) middle cDNA template 0.8ul, 2 × SYBRPremixExTaq5ul, PCRForwardPrimer (10 μ Μ) 0.2ul, PCRReversePrimer (10uM) 0.2ul, ROXReferenceDye (50 ×) 0.2ul, ddH2O3.6ul.Negative control is set.

3) PCR reaction is carried out

Reaction cycle is set to: Holdingstage95 DEG C of 30s; Cyclingstage95 DEG C of 5s, 60 DEG C of 34s, 72 DEG C of 30s, 40cycles; Meltcurvestage95 DEG C of 15s, 60 DEG C of 1min, 95 DEG C of 15s, 40 circulations.

Fig. 3 melting curve represents three pairs of primers (b, c, melting curve design sketch d) reacted with the PCR of the primer (a) in invention respectively.As shown in the figure, (melting curve of b, c, d) PCR reaction does not all have unique melting temperature (Tm) Tm to three pairs of primers, illustrates that primer does not have specificity, there is nonspecific amplification, thus can not be used for the expression level judging gene mRNA.And the melting curve of primer (a) in the present invention has single peak value, illustrate that this primer has good specificity, may be used for the expression level change judging IL-6 factor mRNA, reliable results.

Below the preferred embodiment of the invention is illustrated, but the invention is not limited to described embodiment, those of ordinary skill in the art also can make all equivalent modification or replacement under the prerequisite without prejudice to the invention spirit, and these equivalent modification or replacement are all included in the application's claim limited range.

Claims (6)

1. a naked mole cytokine IL-6 gene PCR detects primer:

Forward primer as shown in SEQIDNO:1,

Reverse primer is as shown in SEQIDNO:2.

2. a naked mole cytokine IL-6 gene PCR detection kit, its detection primer is:

Forward primer: as shown in SEQIDNO:1,

Reverse primer: as shown in SEQIDNO:2.

3. naked mole cytokine IL-6 gene PCR detection kit according to claim 2, also comprises:

2×SYBRPremixExTaq5ul，

50×ROXReferenceDye0.2ul，

ddH ₂O3.6ul。

4. naked mole cytokine IL-6 gene PCR detection kit according to claim 2, comprising:

Forward primer: as shown in SEQIDNO:1,

Reverse primer: as shown in SEQIDNO:2;

2×SYBRPremixExTaq5ul；

50×ROXReferenceDye0.2ul；

ddH ₂O3.6ul。

5. a detection method for naked mole cytokine IL-6 gene, it is characterized in that, described detection method comprises the steps:

Extract total mRNA of naked mole Macrophage Cell, and reverse transcription is cDNA as detection sample, use Real-timePCR method to detect, draw the expression level of IL-6, primer sequence is as follows:

Forward primer: as shown in SEQIDNO:1,

Reverse primer: as shown in SEQIDNO:2.

6. the detection method of naked mole cytokine IL-6 gene according to claim 5, it is characterized in that, concrete detecting step is:

A. mRNA is extracted:

The naked mole scavenger cell of separation and Culture, extract cell total rna, and reverse transcription is cDNA;

B. real-time fluorescence quantitative PCR reaction system 10ul is prepared:

Get cDNA template 0.8ul in steps A, 2 × SYBRPremixExTaq5ul, the PCR reverse primer 0.2ul of the PCR forward primer 0.2ul of 10 μ Μ, 10 μ Μ, 50 × ROXReferenceDye0.2ul, ddH ₂o3.6ul; Negative control is set;

C. PCR reaction is carried out:

Reaction cycle is set to: 95 DEG C of 30s cycles stage of holding stage, 95 DEG C of 5s, 60 DEG C of 34s, 72 DEG C of 30s, 40 circulations; Solubility curve stage 95 DEG C of 15s, 60 DEG C of 1min, 95 DEG C of 15s, 40 circulations.