CN106566833B - Mdl1及其定量检测方法 - Google Patents

Mdl1及其定量检测方法 Download PDF

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CN106566833B
CN106566833B CN201610854317.0A CN201610854317A CN106566833B CN 106566833 B CN106566833 B CN 106566833B CN 201610854317 A CN201610854317 A CN 201610854317A CN 106566833 B CN106566833 B CN 106566833B
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高山
姚雪
卜文俊
阮吉寿
程智
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Abstract

本发明公开了人线粒体基因MDL1和MDL1AS转录的RNA的cDNA序列及其定量检测方法，属于生物技术领域。基因MDL1转录的RNA的cDNA序列为：1)SEQ ID NO.1所示核苷酸序列；或2)SEQ ID NO.1所示核苷酸序列经替换、删除或插入一个或几个核苷酸形成的核苷酸序列。基因MDL1AS转录的RNA的cDNA序列为：1)SEQ ID NO.2所示核苷酸序列；或2)SEQ ID NO.2所示核苷酸序列经替换、删除或插入一个或几个核苷酸形成的核苷酸序列。本发明验证了基因MDL1在多种肿瘤细胞中表达的显著性变化，有非常重要的临床检测价值。

Description

MDL1及其定量检测方法
技术领域
本发明属于生物技术领域，具体涉及两个人线粒体基因MDL1和MDL1AS转录的RNA的cDNA序列及其定量检测方法。
背景技术
动物线粒体进化上极端保守，大多数动物线粒体基因组编码了2个rRNA，13个mRNA和22个tRNA。线粒体是细胞进行有氧呼吸的主要场所，为生命活动提供能量。肿瘤等多种疾病的发生或发展，离不开线粒体基因的异常表达。但是，当前对于线粒体基因表达的基础研究仍然处于初级阶段，缺少突破性进展。直到2016年，南开大学高山和卜文俊等通过全长转录组测序大数据挖掘，在国际上首次发现了两个人线粒体长非编码基因MDL1和MDL1AS，并通过多种实验手段确认其cDNA的全长序列。这两个基因位于一直以来被认为是不发生转录的动物线粒体基因组的控制区(也称作D-Loop区)。接下来的研究发现，含有一个控制区的动物线粒体基因组普遍编码MDL1和MDL1AS。进一步的实验又发现，人MDL1基因在肿瘤等一些线粒体基因表达异常的细胞或组织中有显著性表达变化，因此，该基因有作为临床检测或治疗靶点的可能性。
发明内容
本发明的目的是在国际上首次设计一套简单的基于qRT-PCR的检测方法，检测MDL1基因和MDL1AS基因在各种细胞或组织中的表达量，以支持基础研究和临床应用。
一种人线粒体基因MDL1转录的RNA，其特征在于所述RNA的cDNA序列为
(1)SEQ ID NO.1所示的核苷酸序列；或
(2)SEQ ID NO.1所示核苷酸序列经替换、删除或插入一个或几个核苷酸形成的核苷酸序列。
一种人线粒体基因MDL1AS转录的RNA，其特征在于所述RNA的cDNA序列为
(1)SEQ ID NO.2所示的核苷酸序列；或
(2)SEQ ID NO.2所示核苷酸序列经替换、删除或插入一个或几个核苷酸形成的核苷酸序列。
所述基因MDL1和基因MDL1AS转录的RNA的qRT-PCR检测方法，其特征在于所述RNA的反转引物由20个bp以上的接头序列5′-CTGATCTAGAGGTACCGGATCC-3′与18个Oligo(dT)组成。
所述基因MDL1转录的RNA的qRT-PCR检测方法，其特征在于所述RNA的cDNA扩增需要以下任意一对引物：
(1)引物对1上游5′-CCACAGCACTTAAACACATCTCTGC-3′
引物对1下游5′-CTGATCTAGAGGTACCGGATCC-3′
(2)引物对2上游5′-AACACATCTCTGCCAAACCCCAA-3′
引物对2下游5′-CTGATCTAGAGGTACCGGATCC-3′
(3)引物对3上游5′-AACAAAGAACCCTAACACCAGCC-3′
引物对3下游5′-CTGATCTAGAGGTACCGGATCC-3′
(4)引物对4上游5′-ACACCAGCCTAACCAGATTTC-3′
引物对4下游5′-CTGATCTAGAGGTACCGGATCC-3′
(5)引物对5上游5′-TGCACTTTTAACAGTCACCCC-3′
引物对5下游5′-CTGATCTAGAGGTACCGGATCC-3′
(6)引物对6上游5′-ACACATTATTTTCCCCTCCCACTCC-3′
引物对6下游5′-CTGATCTAGAGGTACCGGATCC-3′
(7)引物对7上游5′-CCCCTCCCACTCCCATACTACTAAT-3′
引物对7下游5′-CTGATCTAGAGGTACCGGATCC-3′
(8)引物对8上游5′-TAACCCCATACCCCGAACCAA-3′
引物对8下游5′-CTGATCTAGAGGTACCGGATCC-3′
(9)引物对9上游5′-TATTTTCCCCTCCCACTCCCATACT-3′
引物对9下游5′-CTGATCTAGAGGTACCGGATCC-3′
(10)引物对10上游5′-AACCCTAACACCAGCCTAACCAG-3′
引物对10下游5′-CTGATCTAGAGGTACCGGATCC-3′
(11)引物对11上游5′-CTTAAACACATCTCTGCCAAACCCC-3′
引物对11下游5′-CTGATCTAGAGGTACCGGATCC-3′
(12)引物对12上游5′-CTCCCACTCCCATACTACTAATC-3′
引物对12下游5′-CTGATCTAGAGGTACCGGATCC-3′
(13)引物对13上游5′-CATACCCCGAACCAACCAA-3′
引物对13下游5′-CTGATCTAGAGGTACCGGATCC-3′
(14)引物对14上游5′-CCGAACCAACCAAACCCCAAA-3′
引物对14下游5′-CTGATCTAGAGGTACCGGATCC-3′
(15)引物对15上游5′-CAACCAAACCCCAAAGACACC-3′
引物对15下游5′-CTGATCTAGAGGTACCGGATCC-3′
(16)引物对16上游5′-TCTCTGCCAAACCCCAAAA-3′
引物对16下游5′_CTGATCTAGAGGTACCGGATCC-3′
(17)引物对17上游5′-GCACTTAAACACATCTCTGCC-3′
引物对17下游5′-CTGATCTAGAGGTACCGGATCC-3′
(18)引物对18上游5′-CTAACACCAGCCTAACCAGAT-3′
引物对18下游5′-CTGATCTAGAGGTACCGGATCC-3′
(19)引物对19上游5′-CGGTATGCACTTTTAACAGTCACCC-3′
引物对19下游5′-CTGATCTAGAGGTACCGGATCC-3′
(20)引物对20上游5′-ACACACACACCGCTGCTAA-3′
引物对20下游5′-CTGATCTAGAGGTACCGGATCC-3′。
所述基因MDL1AS转录的RNA的qRT-PCR检测方法，其特征在于所述RNA的cDNA扩增需要以下任意一对引物：
(1)引物对1上游5′-CTATGTACTGTTAAGGGTGGGTAGG-3′
引物对1下游5′-CTGATCTAGAGGTACCGGATCC-3′
(2)引物对2上游5′-GGTGGCTTTGGAGTTGCAGTT-3′
引物对2下游5′-CTGATCTAGAGGTACCGGATCC-3′
(3)引物对3上游5′-GTTGAGGGTTGATTGCTGTACTTGC-3′
引物对3下游5′-CTGATCTAGAGGTACCGGATCC-3′
(4)引物对4上游5′-GGGGTTTTGATGTGGATTGGGT-3′
引物对4下游5′-CTGATCTAGAGGTACCGGATCC-3′
(5)引物对5上游5′-GGTACCGTACAATATTCATGGTGGC-3′
引物对5下游5′-CTGATCTAGAGGTACCGGATCC-3′
(6)引物对6上游5′-TACATAGCGGTTGTTGATGGGTGAG-3′
引物对6下游5′-CTGATCTAGAGGTACCGGATCC-3′
(7)引物对7上游5′-TGGTACCCAAATCTGCTTCCC-3′
引物对7下游5′-CTGATCTAGAGGTACCGGATCC-3′
(8)引物对8上游5′-GCAGTTGATGTGTGATAGTTGAGGG-3′
引物对8下游5′-CTGATCTAGAGGTACCGGATCC-3′
(9)引物对9上游5′-ACTGTTAAGGGTGGGTAGGTTTG-3′
引物对9下游5′-CTGATCTAGAGGTACCGGATCC-3′
(10)引物对10上游5′-GTAGGTTTGTTGGTATCCTAGTGGG-3′
引物对10下游5′-CTGATCTAGAGGTACCGGATCC-3′。
所述基因MDL1转录的RNA在检测线粒体基因表达异常中的应用。
所述基因MDL1转录的RNA在肿瘤检测中的应用。
所述基因MDL1AS转录的RNA在检测线粒体基因表达异常中的应用。
所述基因MDL1AS转录的RNA在肿瘤检测中的应用。
所述肿瘤，其特征在于包括肺癌、乳腺癌、肝癌、宫颈癌、淋巴癌、前列腺癌和神经母细胞瘤。
本发明的实验证明，根据特殊设计的反转引物，可以从多种肿瘤细胞系中通过qRT-PCR检测到MDL1和MDL1AS基因表达，再通过肿瘤细胞系与正常组织的检测结果对比，发现MDL1基因在肿瘤细胞中表达有显著变化。
附图说明
图1为SH-SY5Y细胞系中的MDL1基因转录的RNA的qRT-PCR产物的电泳图。
图2为Hela细胞系中的MDL1基因转录的RNA的qRT-PCR产物的电泳图。
具体实施方式
以下结合实施例来进一步说明本发明。
实施例
(1)细胞培养：肺癌(NCI-H460)、乳腺癌(MCF-7)、肝癌(HepG2)、宫颈癌(Hela)、淋巴癌(HTL-90)、前列腺癌(PC3)和神经母细胞瘤(SH-SY5Y)细胞系利用含有10％胎牛血清的DMEM培养基培养，每两天更换一次培养基，每四天传代一次。
(2)总RNA提取：参照试剂盒RNAiso Plus(TaKaRa，Japan)提取，共得到体积为25μL，浓度为1.3μg/μL的总RNA。
(3)RNA反转：从总RNA中取出0.8～1μg RNA，参照试剂盒PrimeScript RT ReagentKit with gDNA Eraser(TaKaRa，Japan)进行RNA反转，反转使用了40-nt长度的特殊引物(CTGATCTAGAGGTACCGGATCC-T18)。
(4)cDNA扩增：参照试剂盒SYBR Premix Ex Taq II(TaKaRa，Japan)进行cDNA扩增，使用的qRT-PCR仪器为德国Eppendorf公司生产的Mastercycler ep Realplex2。PCR条件为：95℃初始变性30s，40次PCR循环，每次循环(95℃变性10s，55℃退火10s然后68℃延伸20s)。扩增使用内参基因GAPDH(143-bp)，GAPDH的上下游引物分别为(ACATCGCTCAGACACCATG)和(TGTAGTTGAGGTCAATGAAGGG)。
(5)产物的确认：扩增产物通过凝胶电泳和Sanger测序两种手段进行确认。凝胶电泳使用1％的琼脂糖进行。Sanger测序采用双向测通的方法，由生工生物工程(上海)股份有限公司完成。
(6)实验结果：以内参基因GAPDH作对照，显示了基因MDL1和MDL1AS在多种肿瘤细胞系中高表达；相对正常组织，MDL1基因在肿瘤细胞中表达有显著变化。
[0001] SEQUENCE LISTTNG
[0002] <110>南开大学
[0003] <120> MDL1及其定量检测方法
[0004] <130>
[0005] <160>2
[0006] <170>PatentIn version 3.5
[0007] <210>1
[0008] <211>1162
[0009] <212>DNA
[0010] <213>Homo sapiens
[0011] <400>1
[0012] ttctttcatg gggaagcaga tttgggtacc acccaagtat tgactcacccatcaacaacc 60
[0013] gctatgtatt tcgtacatta ctgccagcca ccatgaatat tgtacggtaccataaatact 120
[0014] tgaccacctg tagtacataa aaacccaatc cacatcaaaa ccccctccccatgcttacaa 180
[0015] gcaagtacag caatcaaccc tcaactatca cacatcaact gcaactccaaagccacccct 240
[0016] cacccactag gataccaaca aacctaccca cccttaacag tacatagtacataaagccat 300
[0017] ttaccgtaca tagcacatta cagtcaaatc ccttctcgtc cccatggatgacccccctca 360
[0018] gataggggtc ccttgaccac catcctccgt gaaatcaata tcccgcacaagagtgctact 420
[0019] ctcctcgctc cgggcccata acacttgggg gtagctaaag tgaactgtatccgacatctg 480
[0020] gttcctactt cagggtcata aagcctaaat agcccacacg ttccccttaaataagacatc 540
[0021] acgatggatc acaggtctat caccctatta accactcacg ggagctctccatgcatttgg 600
[0022] tattttcgtc tggggggtat gcacgcgata gcattgcgag acgctggagccggagcaccc 660
[0023] tatgtcgcag tatctgtctt tgattcctgc ctcatcctat tatttatcgcacctacgttc 720
[0024] aatattacag gcgaacatac ttactaaagt gtgttaatta attaatgcttgtaggacata 780
[0025] ataataacaa ttgaatgtct gcacagccac tttccacaca gacatcataacaaaaaattt 840
[0026] ccaccaaacc ccccctcccc cgcttctggc cacagcactt aaacacatctctgccaaacc 900
[0027] ccaaaaacaa agaaccctaa caccagccta accagatttc aaattttatcttttggcggt 960
[0028] atgcactttt aacagtcacc ccccaactaa cacattattt tcccctcccactcccatact 1020
[0029] actaatctca tcaatacaac ccccgcccat cctacccagc acacacacaccgctgctaac 1080
[0030] cccatacccc gaaccaacca aaccccaaag acacccccca ca 1122
[0031] <210>2
[0032] <211>952
[0033] <212>DNA
[0034] <213>Homo sapiens
[0035] <400>2
[0036] aagataaaat ttgaaatctg gttaggctgg tgttagggtt ctttgtttttggggtttggc 60
[0037] agagatgtgt ttaagtgctg tggccagaag cgggggaggg ggggtttggtggaaattttt 120
[0038] tgttatgatg tctgtgtgga aagtggctgt gcagacattc aattgttattattatgtcct 180
[0039] acaagcatta attaattaac acactttagt aagtatgttc gcctgtaatattgaacgtag 240
[0040] gtgcgataaa taataggatg aggcaggaat caaagacaga tactgcgacatagggtgctc 300
[0041] cggctccagc gtctcgcaat gctatcgcgt gcataccccc cagacgaaaataccaaatgc 360
[0042] atggagagct cccgtgagtg gttaataggg tgatagacct gtgatccatcgtgatgtctt 420
[0043] atttaagggg aacgtgtggg ctatttaggc tttatgaccc tgaagtaggaaccagatgtc 480
[0044] ggatacagtt cactttagct acccccaagt gttatgggcc cggagcgaggagagtagcac 540
[0045] tcttgtgcgg gatattgatt tcacggagga tggtggtcaa gggacccctatctgaggggg 600
[0046] gtcatccatg gggacgagaa gggatttgac tgtaatgtgc tatgtacggtaaatggcttt 660
[0047] atgtactatg tactgttaag ggtgggtagg tttgttggta tcctagtgggtgaggggtgg 720
[0048] ctttggagtt gcagttgatg tgtgatagtt gagggttgat tgctgtacttgcttgtaagc 780
[0049] atggggaggg ggttttgatg tggattgggt ttttatgtac tacaggtggtcaagtattta 840
[0050] tggtaccgta caatattcat ggtggctggc agtaatgtac gaaatacatagcggttgttg 900
[0051] atgggtgagt caatacttgg gtggtaccca aatctgcttc cccatgaaag aa 952

Claims (2)

1.一种人线粒体基因MDL1转录的RNA的检测试剂在制备宫颈癌和神经母细胞瘤检测试剂中的应用，其特征在于所述基因MDL1转录的RNA的cDNA序列为SEQ ID NO.1所示的核苷酸序列。
2.一种人线粒体基因MDL1AS转录的RNA的检测试剂在制备宫颈癌和神经母细胞瘤检测试剂中的应用，其特征在于所述基因MDL1AS转录的RNA的cDNA序列为SEQ ID NO.2所示的核苷酸序列。
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LN881448.1;NCBI;《NCBI》;20160906;第1页 *
NCBI.GU069940.1.《NCBI》.2010,第1页. *
NCBI.LN881448.1.《NCBI》.2016,第1页. *
宫颈癌线粒体DNA D-Loop区基因突变的研究;陈道桢等;《肿瘤》;20050731;第25卷(第4期);摘要 *
线粒体DNA控制区研究进展;郭学光等;《肿瘤防治杂志》;20051130;第12卷(第22期);摘要、第1748页右栏第1-2段 *

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