小鼠骨骼肌纤维类型定量PCR内参基因的筛选

doi:10.13560/j.cnki.biotech.bull.1985.2020-0540

摘要/Abstract

摘要：

旨在筛选定量PCR检测不同骨骼肌纤维类型的稳定内参基因,为骨骼肌的能量和糖代谢等功能研究提供基础数据。试验选用6周龄小鼠,采集腓肠肌（Gastrocnemius muscle,GAS）、比目鱼肌（Soleus,SOL）、胫骨前肌（Tibialis anterior muscle,TA）和趾长伸肌（Extensor digitorum longus,EDL）为试验材料。以荧光定量PCR法检测Gapdh、Actb、Rer1、Hprt1、Ppia、Rpl7、B2m、Sdha和Rpl27等9个内参基因的mRNA水平,并采用Delta CT、geNorm、NormFinder、BestKeeper和RefFinder五种方法对其表达稳定性进行评估。结果显示,Delta CT法分析稳定性前3个基因为Rpl7>Actb>Rer1,以NormFinder法分析稳定性前3个基因是Rpl7>Rer1>Actb,以BestKeeper法分析稳定性前3个基因是Rpl7>Gapdh> Actb,而以geNorm法分析稳定性前3个基因是Actb>B2m>Rpl27。最后通过RefFinder法综合分析Rpl7是最稳定的内参基因,且5种方法中Ppia均最不稳定。因此,Rpl7可作为小鼠骨骼肌纤维类型检测的稳定内参基因。

关键词: 小鼠, 骨骼肌纤维, 内参基因, RT-PCR

Abstract:

The purpose of this study is to screen the internal reference genes stably expressed in different types of skeletal muscle fibers,so as to provide a reliable basic data for the study of energy and glucose metabolism in skeletal muscle. In this experiment,gastrocnemius muscle,soleus,tibialis anterior muscle and extensor digitorum longus were collected from 6-week-old female mice. The mRNA levels of Gapdh,Actb,Rer1,Hprt1,Ppia,Rpl7,B2m,Sdha and Rpl27 were detected by RT-qPCR,and their expression stabilities were evaluated by Delta CT,geNorm,NormFinder,BestKeeper and RefFinder. The results showed that the top three genes of stability analyzed by Delta CT method were Rpl7 > Actb > Rer1. The top three genes of stability analyzed by NormFinder method were Rpl7>Rer1>Actb. The top three genes of stability analyzed by BestKeeper method were Rpl7> Gapdh> Actb,and the top three genes of stability analyzed by geNorm method were Actb>B2m>Rpl27. Finally,Rpl7 was the most stable internal reference gene after comprehensively analyzed by RefFinder method,while Ppia was the most unstable by these methods. Therefore,Rpl7 can be used as a stable internal reference gene for the detection of skeletal muscle fiber types in mouse.

Key words: mouse, skeletal muscle fiber, internal reference gene, RT-PCR

张静, 熊燕, 华永琳, 郭玉, 熊显荣, 字向东, 李键. 小鼠骨骼肌纤维类型定量PCR内参基因的筛选[J]. 生物技术通报, 2021, 37(2): 71-79.

ZHANG Jing, XIONG Yan, HUA Yong-lin, GUO Yu, XIONG Xian-rong, ZI Xiang-dong, LI Jian. Screening of Reference Genes for Quantitative PCR of Skeletal Muscle Fiber Types in Mice[J]. Biotechnology Bulletin, 2021, 37(2): 71-79.

图/表 7

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基因	登陆号	引物序列（5'-3'）	产物长度/bp
Gapdh	NM_008084.3	F：AGGTCGGTGTGAACGGATTTG R：TGTAGACCATGTAGTTGAGGTCA	123
Actb	NM_007393.5	F：GCCTCACTGTCCACCTTCCA R：GGGCCGGACTCATCGTACT	62
Hprt1	NM_013556.2	F：AATTATGGACAGGACTGAACGTCTTGCT R：TCCAGCAGGTCAGCAAAGAATTTATAGC	117
Ppia	NM_008907.2	F：GCCAGGGTGGTGACTTTA R：AACTGGGAACCGTTTGTG	151
Rer1	NM_026395.2	F：GCCTTGGGAATTTACCACCT R：CTTCGAATGAAGGGACGAAA	137
Rpl7	NM_025433.3	F：ACGGTGGAGCCTTATGTGAC R：TCCGTCAGAGGGACTGTCTT	110
B2m	NM_009735.3	F：GAAGCCGAACATACTGAACTG R：CACATGTCTCGATCCCAGTAG	246
Sdha	NM_023281.1	F：GCGATATGACACCAGTTATTT R：TTGCTCTTATTCGGTGTATGGG	122
Rpl27	NM_011289.3	F：AAGCCGTCATCGTGAAGAACA R：CTTGATCTTGGATCGCTTGGC	143
Myh2	NM_001039545.2	F：AAGCGAAGAGTAAGGCTGTC R：CTTGCAAAGGAACTTGGGCTC	136
Myh4	NM_010855.3	F：GAAGAGCCGAGAGGTTCACAC R：CAGGACAGTGACAAAGAACGTC	108
Myh1	NM_030679.2	F：GAAGAGTGATTGATCCAAGTG R：TATCTCCCAAAGTTATGAGTACA	98

基因	登陆号	引物序列（5'-3'）	产物长度/bp
Gapdh	NM_008084.3	F：AGGTCGGTGTGAACGGATTTG R：TGTAGACCATGTAGTTGAGGTCA	123
Actb	NM_007393.5	F：GCCTCACTGTCCACCTTCCA R：GGGCCGGACTCATCGTACT	62
Hprt1	NM_013556.2	F：AATTATGGACAGGACTGAACGTCTTGCT R：TCCAGCAGGTCAGCAAAGAATTTATAGC	117
Ppia	NM_008907.2	F：GCCAGGGTGGTGACTTTA R：AACTGGGAACCGTTTGTG	151
Rer1	NM_026395.2	F：GCCTTGGGAATTTACCACCT R：CTTCGAATGAAGGGACGAAA	137
Rpl7	NM_025433.3	F：ACGGTGGAGCCTTATGTGAC R：TCCGTCAGAGGGACTGTCTT	110
B2m	NM_009735.3	F：GAAGCCGAACATACTGAACTG R：CACATGTCTCGATCCCAGTAG	246
Sdha	NM_023281.1	F：GCGATATGACACCAGTTATTT R：TTGCTCTTATTCGGTGTATGGG	122
Rpl27	NM_011289.3	F：AAGCCGTCATCGTGAAGAACA R：CTTGATCTTGGATCGCTTGGC	143
Myh2	NM_001039545.2	F：AAGCGAAGAGTAAGGCTGTC R：CTTGCAAAGGAACTTGGGCTC	136
Myh4	NM_010855.3	F：GAAGAGCCGAGAGGTTCACAC R：CAGGACAGTGACAAAGAACGTC	108
Myh1	NM_030679.2	F：GAAGAGTGATTGATCCAAGTG R：TATCTCCCAAAGTTATGAGTACA	98

基因	标准曲线方程	斜率	扩增效率/％	决定系数R²
Gapdh	y = -3.2786x + 32.42	-3.278 6	101.8	0.994 5
Actb	y = -3.3303x + 38.42	-3.330 3	99.7	0.992 7
Hprt1	y = -3.3956x + 41.68	-3.395 6	97.0	0.996 5
Ppia	y = -3.4648x + 42.49	-3.464 8	94.4	0.982 6
Rer1	y = -3.0843x + 37.46	-3.084 3	111.0	0.992 8
Rpl7	y = -3.0167x + 38.19	-3.016 7	114.5	0.992 6
B2m	y = -3.0289x + 18.30	-3.028 9	113.9	0.990 7
Sdha	y = -3.1061x + 38.727	-3.106 1	110.0	0.986 0
Rpl27	y = -3.346x + 37.30	-3.346 0	99.0	0.990 8

基因	标准曲线方程	斜率	扩增效率/％	决定系数R²
Gapdh	y = -3.2786x + 32.42	-3.278 6	101.8	0.994 5
Actb	y = -3.3303x + 38.42	-3.330 3	99.7	0.992 7
Hprt1	y = -3.3956x + 41.68	-3.395 6	97.0	0.996 5
Ppia	y = -3.4648x + 42.49	-3.464 8	94.4	0.982 6
Rer1	y = -3.0843x + 37.46	-3.084 3	111.0	0.992 8
Rpl7	y = -3.0167x + 38.19	-3.016 7	114.5	0.992 6
B2m	y = -3.0289x + 18.30	-3.028 9	113.9	0.990 7
Sdha	y = -3.1061x + 38.727	-3.106 1	110.0	0.986 0
Rpl27	y = -3.346x + 37.30	-3.346 0	99.0	0.990 8

Genes	Delta CT	geNorm	NormFinder	BestKeeper	RefFinder
Actb	1.11（2）	0.49（1）	0.54（3）	0.53（3）	2.21（2）
Gapdh	1.46（7）	0.80（7）	1.13（8）	0.46（2）	5.29（6）
Ppia	3.33（9）	1.49（9）	3.26（9）	2.92（9）	9.00（9）
B2m	1.19（4）	0.49（2）	0.72（5）	0.55（5）	3.16（3）
Hprt1	1.63（8）	0.97（8）	1.09（7）	1.32（8）	7.74（8）
Sdha	1.30（6）	0.71（6）	0.73（6）	0.86（7）	6.24（7）
Rpl7	1.07（1）	0.58（4）	0.16（1）	0.45（1）	1.41（1）
Rpl27	1.20（5）	0.53（3）	0.71（4）	0.53（4）	3.66（5）
Rer1	1.12（3）	0.64（5）	0.30（2）	0.69（6）	3.66（4）