CN105543227A - Chicken enteritis salmonella infection resistance character molecular marker gga-miR-1662 and detection method thereof - Google Patents

Chicken enteritis salmonella infection resistance character molecular marker gga-miR-1662 and detection method thereof Download PDF

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CN105543227A
CN105543227A CN 201610015399 CN201610015399A CN105543227A CN 105543227 A CN105543227 A CN 105543227A CN 201610015399 CN201610015399 CN 201610015399 CN 201610015399 A CN201610015399 A CN 201610015399A CN 105543227 A CN105543227 A CN 105543227A
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李显耀
吴桂贤
刘丽英
刘肖意
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山东农业大学
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Abstract

The invention relates to the technical field of gene engineering, and provides a chicken enteritis salmonella infection resistance character molecular marker gga-miR-1662 and a detection method thereof. The resistance molecular marker is chicken miRNA gga-miR-1662. The inventor finds through research that enteritis salmonella infection of chickens can be regulated and controlled through gga-miR-1662 by regulating and controlling expression of the gene TLR1LA, and therefore gga-miR-1662 can be used as the molecular marker of enteritis salmonella infection resistance detection, the inventor further provides the detection method of the marker, and therefore the foundation is laid for disease-resistant genetic breeding of chickens.

Description

-种鸡肠炎沙口氏菌感染抗性性状分子标巧gga-m i R-1662及其检测方法 - enteritis Shakou breeder's infection resistance trait molecular markers Qiao gga-m i R-1662 and its detection method

技术领域 FIELD

[0001] 本发明设及基因工程技术领域,提供了一种鸡肠炎沙口氏菌感染抗性分子标记gga-miR-1662及其检测方法。 [0001] Technical Field The present invention is provided and genetic engineering, there is provided a molecule chickens infected by Salmonella enteritidis Shakou tag gga-miR-1662 and its detection method.

背景技术 Background technique

[0002] 肠炎沙口氏菌(Salmonella enteritidis)为革兰氏阴性菌,是世界公认的食源性致病菌之一,不仅对蛋鸡生产造成重大影响,而且它能通过家禽副产品给人类的健康带来很大的威胁。 [0002] Shakou Salmonella enteritidis (Salmonella enteritidis) Gram-negative bacteria, is recognized worldwide as one of foodborne pathogens, not only have a significant impact on the egg production, but it can give humans through poultry by-products great health threat. 运种病菌主要是通过动物性产品包括禽肉,鸡蛋和奶类及奶制品等导致人中毒甚至死亡。 Yun kinds of germs mainly through animal products, including poultry meat, eggs and milk and milk products as a result of poisoning or even death. 因此如何获得抗性高的遗传个体成为亟待解决的问题之一。 So how to get individual high genetic resistance to become one of the problems to be solved.

[0003] 小RNA(miRNA)是一类长度约22nt的内源性非编码小RNA,与祀基因3'UTR(非翻译区)结合在转录后水平调控祀基因的表达,在细胞增殖、分化、调亡、神经发育、脂肪代谢等多个生物学过程中发挥重要作用。 [0003] Small RNA (miRNA) are a class of endogenous length non-coding RNA of about 22nt, and Si gene 3'UTR (untranslated region) incorporated in the regulation of the expression level of post-transcriptional gene worship, cell proliferation, differentiation , apoptosis, more biological neural development, fat metabolism play an important role.

发明内容 SUMMARY

[0004] 本发明的发明人针对上述现有技术的情况,结合长期研究和探索,提供了一种鸡肠炎沙口氏菌感染抗性分子标记gga-miR-1662及其检测方法,该抗性分子标记为鸡miRNA gga-miR-1662,发明人经过研究发现,gga-miR-1662可W通过调控TLR1LA基因的表达调控肠炎沙口氏菌对鸡的感染,因此gga-miR-1662可W作为肠炎沙口氏菌感染抗性检测的分子标记,发明人进一步提供了其检测方法,从而为鸡的抗病遗传育种奠定基础。 [0004] The inventors of the present invention for the case of the above-described prior art, in conjunction with long-term research and exploration, a molecular marker of infection resistance gga-miR-1662, and a detection method Shakou coli enteritis chicken, this resistance marker chicken miRNA gga-miR-1662, the inventor has found that, gga-miR-1662 may be W regulation infection colitis Shakou coli in chickens by regulating the expression TLR1LA gene, thus gga-miR-1662 can be W as Salmonella enteritidis molecular Shakou infected by a detectable label, the inventors have further provided a detection method, thereby laying the foundation for resistance genetic breeding chickens.

[0005] 发明人提供的具体技术方案如下: [0005] DETAILED aspect inventors are as follows:

[0006] 发明人首先提供了一种鸡肠炎沙口氏菌感染抗性分子标记,该抗性分子标记为鸡miRNA gga-miR-1662,其核巧酸序列如沈Q ID NO: 1所示; [0006] The inventors have first provided a molecular marker one kind of chicken infected by Shakou Salmonella enteritidis, as the resistance marker chicken miRNA gga-miR-1662, its nuclear clever acid sequence as Shen Q ID NO: 1 shown in FIG. ;

[0007] 发明人进一步给出了该分子标记的检测方法如下: [0007] The inventors have further given molecular marker detection method is as follows:

[000引gga-miR-1662的表达量检测 Expression gga-miR-1662 of [primers detected 000

[0009] miRNA反转录 [0009] miRNA reverse transcription

[0010] 用One St邱PivimeS.cript麽miRNA cDNA Synthesis Kit(Perfect Real Time)试剂盒,严格按说明书进行。 [0010] One St Qiu PivimeS.cript with it miRNA cDNA Synthesis Kit (Perfect Real Time) kit, in strict accordance with the instructions. 反转录体系为20化(具体见表1)。 20 of the reverse transcription system (see Table 1). 反应程序为:37°C,60min;85°C, 5s;反应完成后获得的cDNA置于-20°C保存备用; The reaction program was: 37 ° C, 60min; 85 ° C, 5s; cDNA obtained after completion of the reaction was placed at -20 ° C for later use;

[0011] 表1 miRNA反转录体系(20yL) [0011] Table 1 miRNA reverse transcription system (20yL)

[0012] [0012]

Figure CN105543227AD00031

[0013]其中所述的盲肠总RNA为鸡盲肠总RNA,采用常规方法获得, [0013] Total RNA wherein the caecum caecum is total RNA, using conventional methods,

[0014] gga-miR-1662 巧光定量PCR [0014] gga-miR-1662 light quantitative PCR Qiao

[0015] 根据gga-miR-1662成熟序列设计引物(表2),由上海生工生物工程有限公司合成。 [0015] The gga-miR-1662 mature sequence primers were designed (Table 2), from Shanghai Sangon Biological Engineering Limited synthesis. 利用所设引物,采用SYBR Green I染料法,按照TaKaRa公司的SYBR PrimeScriptTM miRNA RT-PCR Kit说明书,在Stratagene MX3000P巧光定量PCR仪上进行扩增,反应体系如表3所/J、- 〇 Using the primer set, using SYBR Green I dye method, in accordance with TaKaRa company SYBR PrimeScriptTM miRNA RT-PCR Kit instructions, Stratagene MX3000P amplification on the PCR instrument light Qiao, the reaction system as shown in Table 3 / J, - square

[0016] 表2 miRNA定量检测引物 [0016] Table 2 miRNA quantitative detection primer

[0017] [0017]

Figure CN105543227AD00041

[0019] 其中〉gga-miR-1662和U6分别作为Forward qPCR Primer,而Uni-miR qPCR Primer选用试剂盒中自带引物; [0019] wherein> gga-miR-1662, and U6, respectively, as the Forward qPCR Primer, and Uni-miR qPCR Primer selection kit comes with a primer;

[0020] 巧光定量PCR反应程序采用两步法:95°C预变性30s,1个循环;95°C5s,60°C30s,40 个循环;最后添加溶解曲线(95°CImin,62°C30s,95°C30s),1个循环,每个cDNA样品重复Ξ 次。 [0020] Qiao light quantitative PCR reaction procedure step method: 95 ° C denaturation 30s, 1 cycle; 95 ° C5s, 60 ° C30s, 40 cycles; final dissolution profile was added (95 ° CImin, 62 ° C30s, 95 ° C30s), 1 cycle, was repeated for each cDNA sample Ξ times. WU6作为内参基因,用方法来计算miRNA的相对表达量,用SAS8.1软件中单因素方差分析对miRNA表达量差异进行分析。 WU6 gene as internal control, to calculate the relative expression of miRNA method, using one-way ANOVA analysis software SAS8.1 difference of miRNA expression analysis.

[0021] 表3巧光定量PCR体系(2化L) [0021] Table 3 Quantitative PCR System Qiao light (of 2 L)

[0022] [0022]

Figure CN105543227AD00042

[0023] 发明人为了验证上述gga-miR-1662的功能采用了祀基因检测的方式,利用TargetScan及miRanda等软件预测到TLR1LA是gga-miR-1662的祀基因。 [0023] In order to verify the above inventors gga-miR-1662 using a functional genetic testing mode Si, using software miRanda TargetScan and is predicted to worship TLR1LA gene gga-miR-1662's. 结果发现通过分别比较肠炎沙口氏菌感染组与未感染组中miRNA与祀基因的调控方向,可知gga-miR-1662在感染组中显著下调,祀基因化R1LA在感染组中的表达量显著高于未感染组(如图1所示),即gga-miR-1662与化R1LA调控方向相反,表现出相互作用关系。 The results found were compared control the direction enteritis Shakou Salmonella infected and uninfected group of miRNA worship gene found gga-miR-1662 significantly downregulated in the infected group, the expression of worship gene of R1LA infected group was significantly higher than the uninfected group (Figure 1), i.e. gga-miR-1662 and regulation of R1LA opposite direction, showing the interaction between. 说明gga-miR-1662可W通过调控化R1LA基因的表达调控肠炎沙口氏菌对鸡的感染。 Description gga-miR-1662 W can be regulated enteritis Shakou coli infection in chickens by regulating the expression of genes R1LA. 因此gga-miR-1662可W作为肠炎沙口氏菌感染抗性检测的分子标记。 Thus gga-miR-1662 W can be used as molecular Shakou Salmonella enteritidis infected by a detectable label.

[0024] 综上所述,本发明提供了一种鸡肠炎沙口氏菌感染抗性分子标记及其检测方法, 该抗性分子标记为鸡gga-miR-1662,发明人经过研究发现,gga-miR-1662可W通过调控化R1LA基因的表达调控肠炎沙口氏菌对鸡的感染,因此gga-miR-1662可W作为肠炎沙口氏菌感染抗性检测的分子标记,发明人进一步提供了其检测方法,从而为鸡的抗病遗传育种奠定基础。 [0024] In summary, the present invention provides a method for detecting resistance marker and one kind of chicken infected with Salmonella enteritidis sand mouth, the resistance marker chicken gga-miR-1662, the inventor has found that, GGA -miR-1662 W can be regulated Salmonella infection Shakou enteritis in chickens by gene expression and regulation of R1LA therefore gga-miR-1662 can be used as molecular markers W Shakou Salmonella enteritidis infection resistance testing, the inventors provide further its detection methods, thereby laying the foundation for genetic breeding disease-resistant chickens.

附图说明 BRIEF DESCRIPTION

[00巧]图1为gga-miR-1662与祀基因化RILA在感染组相对未感染组中的表达差异倍数柱状图, [Qiao 00] FIG. 1 is a gga-miR-1662 gene and Si RILA histogram of infection fold differential expression relative to non-infected group,

[00%]由图可知,gga-miR-1662在感染组中显著下调,TLR1LA在感染组中的表达量显著高于未感染组,即gga-miR-1662与TLR1LA调控方向相反,表现出相互作用关系。 [00%] is apparent from FIG, gga-miR-1662 significantly reduced in infected group, TLR1LA expression in the infected group was significantly higher than the uninfected group, i.e. gga-miR-1662 and TLR1LA regulation opposite direction, showing another relations role. 说明gga- miR-1662可W通过调控TLR1LA基因的表达调控肠炎沙口氏菌对鸡的感染。 Description gga- miR-1662 W can be regulated enteritis Shakou coli infection in chickens TLR1LA by regulating the expression of genes.

具体实施方式 Detailed ways

[0027] 下述实施例中除特殊说明之外,所采用的均为本领域现有技术; [0027] are examples of the art in addition to special instructions, employed in the following examples;

[0028] 实施例1 [0028] Example 1

[0029] gga-miR-1662的表达量检测 [0029] The expression gga-miR-1662 detection

[0030] miRNA 反转录 [0030] miRNA reverse transcription

[0031 ]用One St邱'PrlmeSc.ri:pt'®miRNA cDNA Synthesis Kit(F*e;rfect Real Time)试剂盒,严格按说明书进行。 [0031] One St by Qiu 'PrlmeSc.ri: pt'®miRNA cDNA Synthesis Kit (F * e; rfect Real Time), strictly according to the instructions for the kit. 反转录体系为20化(表1)。 20 of the reverse transcription system (Table 1). 反应程序为:37°C,60min; 85°C,5s;反应完成后获得的cDNA置于-20°C保存备用; The reaction program was: 37 ° C, 60min; 85 ° C, 5s; cDNA obtained after completion of the reaction was placed at -20 ° C for later use;

[0032] 表1 miRNA反转录体系(20yL) Γ00331 [0032] Table 1 miRNA reverse transcription system (20yL) Γ00331

Figure CN105543227AD00051

[0034] 其中所述的盲肠总RNA为鸡盲肠总RNA,采用常规方法获得, [0034] Total RNA wherein the caecum caecum is total RNA, using conventional methods,

[0035] gga-miR-1662 巧光定量PCR [0035] gga-miR-1662 light quantitative PCR Qiao

[0036] 根据gga-miR-1662成熟序列设计引物(表2),由上海生工生物工程有限公司合成。 [0036] The gga-miR-1662 mature sequence primers were designed (Table 2), from Shanghai Sangon Biological Engineering Limited synthesis. 利用所设引物,采用SYBR Green I染料法,按照TaKaRa公司的SYBR PrimeScriptTM miRNA RT-PCR Kit说明书,在Stratagene MX3000P巧光定量PCR仪上进行扩增,反应体系如表3所/J、- 〇 Using the primer set, using SYBR Green I dye method, in accordance with TaKaRa company SYBR PrimeScriptTM miRNA RT-PCR Kit instructions, Stratagene MX3000P amplification on the PCR instrument light Qiao, the reaction system as shown in Table 3 / J, - square

[0037] 表2 miRNA定量检测引物[00;3 引 [0037] Table 2 miRNA quantitative detection primer [00; 3 primer

Figure CN105543227AD00052

[0039] 其中〉gga-miR-1662和U6分别作为Forward qPCR Primer,而Uni-miR qPCR Primer选用试剂盒中自带引物; [0039] wherein> gga-miR-1662, and U6, respectively, as the Forward qPCR Primer, and Uni-miR qPCR Primer selection kit comes with a primer;

[0040] 巧光定量PCR反应程序采用两步法:95°C预变性30s,1个循环;95°C5s,60°C30s,40 个循环;最后添加溶解曲线(95°CImin,62°C30s,95°C30s),1个循环,每个cDNA样品重复Ξ 次。 [0040] Qiao light quantitative PCR reaction procedure step method: 95 ° C denaturation 30s, 1 cycle; 95 ° C5s, 60 ° C30s, 40 cycles; final dissolution profile was added (95 ° CImin, 62 ° C30s, 95 ° C30s), 1 cycle, was repeated for each cDNA sample Ξ times. WU6作为内参基因,用方法来计算miRNA的相对表达量,用SAS8.1软件中单因素方差分析对miRNA表达量差异进行分析。 WU6 gene as internal control, to calculate the relative expression of miRNA method, using one-way ANOVA analysis software SAS8.1 difference of miRNA expression analysis.

[0041 ] 表3巧光定量PCR体系(20μυ [0041] Table 3 Quantitative PCR clever optical system (20μυ

[0042] [0042]

Figure CN105543227AD00061

[0043] 实施例2 [0043] Example 2

[0044] 祀基因TLRILA的表达量检测 [0044] Gene expression of worship detecting TLRILA

[0045] mRNA反转录 [0045] mRNA by reverse transcription

[0046] 根据试剂盒说明书要求,利用TaKaRa Primer ScriptTM RT reagent kit (Perfect Real Time)试剂盒将mRNA反转录为cDNA,放在-20°C保存备用。 [0046] The kit of the specification requirements using TaKaRa Primer ScriptTM RT reagent kit (Perfect Real Time) mRNA reverse transcription kit of the cDNA, placed at -20 ° C for use. 反转录体系如表4 所示。 Reverse transcription system shown in Table 4. 反转录步骤:37°C,15min;85°C,5s。 Reverse transcription step: 37 ° C, 15min; 85 ° C, 5s.

[0047] 表4反转录体系[004引 [0047] Table 4 Reverse Transcription System [004 primer

Figure CN105543227AD00062

[0049] TLR1LA 巧光定量PCR [0049] TLR1LA Qiao light quantitative PCR

[00加]根据NCBI数据库mRNA序列(登录号:NM_001007488),用Primer Premie巧.0设计巧光定量PCR引物(表5所示),引物由上海生工生物工程有限公司合成。 [Plus 00] The mRNA sequence NCBI database (accession number: NM_001007488), 0.01 skillfully designed using Primer Premie clever light Quantitative PCR primers (Table 5), a primer Co. Shanghai Sangon synthesis.

[0051]表5引物序列 [0051] Table 5 primer sequence

[0化2] [0 of 2]

Figure CN105543227AD00063

[0053] 采用SYBR Green I染料法,按照TaKaRa公司的SY服Premix Ex TaqTM说明书,在Stratagene MX3000P巧光定量PCR仪上进行扩增,反应体系为20化(表6):SYBR Primer Ex TaqTM(2 X)ΙΟμΙ,Forward Primer(10μΜ)0.4μ1,Reverse Primer(10μΜ)0.4μ1,ROX Ref erence Dye Π (50 X )0.4化,cDNA模板化L,灭菌双蒸水(d地2〇)6.祉L,终体积20化。 [0053] The SYBR Green I dye method, in accordance with the service (TaKaRa) SY Premix Ex TaqTM instructions, Stratagene MX3000P amplification on the optical Qiao quantitative PCR reaction system of 20 (Table 6): SYBR Primer Ex TaqTM (2 X) ΙΟμΙ, Forward Primer (10μΜ) 0.4μ1, Reverse Primer (10μΜ) 0.4μ1, ROX Ref erence Dye Π (50 X) 0.4 technology, cDNA template of L, sterile double-distilled water (d ground 2〇) 6. Edwin L, final volume of 20. 反应程序为两步法:95°C预变性30s,PCR反应为:95°C,5s; 60°C,30s;循环40次;添加溶解曲线:95°C,Imin;ere,30s;95°C,30s;每个样品重复3次。 The reaction procedure is a two step process: 95 ° C denaturation 30s, PCR reaction: 95 ° C, 5s; 60 ° C, 30s; 40 cycles; add dissolution profile: 95 ° C, Imin; ere, 30s; 95 ° C, 30s; each sample was repeated 3 times. Wf3-act in作为内参,用2^AGt方法来计算mRNA的相对表达量,用SAS8.1软件中单因素方差分析对mRNA表达量差异进行分析。 Wf3-act in as internal control, the relative expression by 2 ^ AGt method to calculate the mRNA using one-way ANOVA analysis software SAS8.1 difference in the amount of mRNA expression analysis.

[0054] 表6巧光定量PCR体系 [0054] Table 6 clever light quantitative PCR System

[0化5] [0 for 5]

Figure CN105543227AD00071

[0056] 实施例3 [0056] Example 3

[0057]鸡接种肠炎沙口氏菌试验 [0057] chickens inoculated test Salmonella enteritidis Shakou

[005引将沙口氏菌阴性白来航蛋鸡分为感染组与未感染组,感染组每只鸡接种5.8 X 108CRJ肠炎沙口氏菌,未感染组接种憐酸盐缓冲液(PBS),接种后第7天采集盲肠组织样品。 [005 Shakou lead to negative Salmonella White Leghorn into infected and non-infected group, infection per bird were inoculated 5.8 X 108CRJ Shakou Salmonella enteritidis, non-infected group received Rei formate buffer (PBS), 7 days after inoculation cecal tissue samples collected. 按照使用说明,用Trizol法提取盲肠总RNA。 Accordance with the instructions, cecum Total RNA was extracted using Trizol method.

[0059] gga-miR-1662与肠炎沙口氏菌感染的相关性分析 [0059] correlation analysis gga-miR-1662 and enteritis Shakou coli infection

[0060] 利用实施例1所述方法,检测肠炎沙口氏菌感染后盲肠组织gga-miR-1662表达变化。 [0060] Using the procedure described in Example 1, the detection cecal tissue gga-miR-1662 expression after infection of Salmonella enteritidis Shakou. 定量结果显示,gga-miR-1662在感染组盲肠中的表达量显著低于未感染组(Fold change = -2.44)。 The results show the quantitative expression of gga-miR-1662 infection in the cecum was significantly lower than non-infected group (Fold change = -2.44). 说明gga-miR-1662与肠炎沙口氏菌感染具有较强的相关性。 Description gga-miR-1662 and enteritis Shakou coli infection have a strong correlation.

[0061 ] TLR1LA与肠炎沙口氏菌感染的相关性分析 [0061] TLR1LA correlation analysis between the mouth of Salmonella enteritidis infection in sand

[0062] 利用实施例2所述方法,检测盲肠化R1LA表达与肠炎沙口氏菌感染的相关性。 The method of Example 2, the detection of the cecum and bowel R1LA expression Shakou Salmonella Infection by [0062] using embodiments. 结果显示,感染组化R1LA的表达量显著高于未感染组,是未感染组的1.55倍(P<0.05)。 The results showed that the expression of the amount of infection was significantly higher than R1LA uninfected group was 1.55 times (P <0.05) in the non-infected group. 说明TLR1LA与鸡肠炎沙口氏菌感染相关。 Description TLR1LA infection associated with chicken Shakou Salmonella enteritidis.

[0063] gga-miR-1662与其祀基因TLR1LA的互作分析 [0063] Interaction Analysis gga-miR-1662 and its gene TLR1LA of worship

[0064] 通过分别比较感染组与未感染组中miRNA与祀基因的调控方向,可知gga-miR- 1662在感染组中显著下调,TLR1LA在感染组中的表达量显著高于未感染组(如图1),即gga- miR-1662与化R1LA调控方向相反,表现出相互作用关系。 [0064] By respectively comparing infected and uninfected group control the direction of miRNA worship gene found gga-miR- 1662 significantly downregulated in the infected group, the expression in infected group TLR1LA significantly higher than the uninfected group (e.g. FIG. 1), i.e. gga- miR-1662 and regulation of R1LA opposite direction, showing the interaction between. 说明gga-miR-1662可W通过调控化R1LA基因的表达调控肠炎沙口氏菌对鸡的感染。 Description gga-miR-1662 W can be regulated enteritis Shakou coli infection in chickens by regulating the expression of genes R1LA. 因此gga-miR-1662可W作为肠炎沙口氏菌感染抗性检测的分子标记。 Thus gga-miR-1662 W can be used as molecular Shakou Salmonella enteritidis infected by a detectable label.

Claims (2)

1. 一种鸡肠炎沙口氏菌感染抗性分子标记,该抗性分子标记为鸡miRNA gga-miR- 1662,其核巧酸序列如沈Q ID NO: 1所示。 A chicken infected with Salmonella enteritidis Shakou resistance marker, the resistance marker chicken miRNA gga-miR- 1662, such as its nuclear clever acid sequence Shen Q ID NO: 1 shown in FIG.
2. 权利要求1所述鸡肠炎沙口氏菌感染抗性分子标记检测方法,具体步骤如下: gga-miR-1662的表达量检测miRNA反转录用One St邱时.imeScri.p:愧'miRNA cDNA Synthesis Kit(Perfect Real Time)试剂盒, 严格按说明书进行;反转录体系为20化,如表1所示;反应程序为:37°C,60min; 85°C,5s;反应完成后获得的cDNA置于-20°C保存备用; 表1 miRNA反转录体系 When .imeScri.p gga-miR-1662 of detecting miRNA expression inversion recruitment One St Qiu:: 1 2. Chicken Salmonella enteritidis Shakou claim infection resistance marker detection method, the following steps shame ' miRNA cDNA Synthesis kit (Perfect Real Time) kit, in strict accordance with the instructions; reverse transcription of the system 20, as shown in table 1; for the reaction sequence: 37 ° 60min C,; 85 ° C, 5s; after completion of the reaction cDNA obtained was placed at -20 ° C for later use; table 1 miRNA reverse transcription system
Figure CN105543227AC00021
gga-miR-1662 巧光定量PCR 根据gga-miR-1662成熟序列设计引物,如表2所示;利用所设引物,采用SYBR Green I 染料法,按照TaKaRa公司的SYBR PrimeScriptTM miRNA RT-PCR Kit说明书,在Stra化gene MX3000P巧光定量PCR仪上进行扩增,反应体系如表3所示; 表2 miRNA定量检测引物 gga-miR-1662 Qiao light quantitative PCR according gga-miR-1662 mature sequence primers were designed, as shown in Table 2; using the set of primers, using SYBR Green I dye method, in accordance with TaKaRa company SYBR PrimeScriptTM miRNA RT-PCR Kit Manual , amplification of gene MX3000P on Stra clever light quantitative PCR reaction system as shown in table 3; table 2 miRNA quantitative detection primer
Figure CN105543227AC00022
巧光定量PCR反应程序采用两步法:95°C预变性30s,1个循环;95°C5s,60°C30s,40个循环;最后添加溶解曲线(95°(31111111,62°03〇3,95°03〇3),1个循环,每个〇0臟样品重复^次;^ U6作为内参基因,用方法来计算miRNA的相对表达量,用SAS8.1软件中单因素方差分析对gga-miR-1662表达量差异进行分析。 Qiao light quantitative PCR reaction procedure step method: 95 ° C denaturation 30s, 1 cycle; 95 ° C5s, 60 ° C30s, 40 cycles; final dissolution profile was added (95 ° (31111111,62 ° 03〇3, 03〇3 95 °), a cycle, each sample was repeated ^ 〇0 dirty times; ^ U6 gene as internal control, a method to calculate the relative expression levels of miRNA, for analysis by ANOVA gga- software SAS8.1 The expression of miR 1662-difference analysis.
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