Screening of Reference Genes for Quantitative PCR of Skeletal Muscle Fiber Types in Mice

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

Abstract

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

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.

Figures/Tables 7

References 35

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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）