Selection of optimal reference genes for quantitative RT-PCR studies of boar spermatozoa cryopreservation.
Reference genes can be used to normalize mRNA levels across different samples for the exact comparison of the mRNA expression level. It is important to select reference genes with high quality for the accurate interpretation of qRT-PCR data. Although several studies have attempted to validate reference genes in pigs, no validation studies have been performed on spermatozoa samples frozen with different cryoprotectants. In this study, 11 commonly used reference genes (ACTB, B2M, GAPDH, HPRT1, RPL4, SDHA, YWHAZ, PPIA, PGK1, S18, and BLM) were investigated in boar spermatozoa frozen with six different cryoprotectants using qRT-PCR. The expression stability of these reference genes in different samples was evaluated using geNorm (qbase(plus) software), NormFinder, and BestKeeper. The geNorm results revealed that PGK1, ACTB, and RPL4 exhibit high expression stability in all of the samples, and the NormFinder results indicated that GAPDH is the most stable gene. Furthermore, the BestKeeper results indicated that the three most stable genes are PPIA, GAPDH, and RPL4 and that S18, B2M and BLM are the three least stable genes. There are a number of differences in the ranking order of the reference genes obtained using the different algorithms. In conclusion, GAPDH, RPL4, and PPIA were the three most stable genes in frozen boar spermatozoa, as determined based on the cycle threshold coefficient of variation (Ct CV%) and the comprehensive ranking order, and this finding is consistent with the BestKeeper results.
College of Animal Science and Technology, Sichuan Agricultural University, Ya'an, Sichuan 625014, PR China. Electronic address: zengchj@sicau.edu.cn.
Changjun
C
Zeng
College of Animal Science and Technology, Sichuan Agricultural University, Ya'an, Sichuan 625014, PR China.
Lian
L
He
College of Animal Science and Technology, Sichuan Agricultural University, Ya'an, Sichuan 625014, PR China.
Wenpei
W
Peng
College of Animal Science and Technology, Sichuan Agricultural University, Ya'an, Sichuan 625014, PR China.
Li
L
Ding
College of Animal Science and Technology, Sichuan Agricultural University, Ya'an, Sichuan 625014, PR China.
Keyi
K
Tang
College of Animal Science and Technology, Sichuan Agricultural University, Ya'an, Sichuan 625014, PR China.
Donghui
D
Fang
College of Animal Science and Technology, Sichuan Agricultural University, Ya'an, Sichuan 625014, PR China.
Yan
Y
Zhang
D000818:Animals; D015925:Cryopreservation; D003451:Cryoprotective Agents / Q000494:pharmacology*; D015870:Gene Expression / Q000187:drug effects*; D020043:Genes, Essential; D024581:Glyceraldehyde-3-Phosphate Dehydrogenase (Phosphorylating) / Q000235:genetics; D008297:Male; D019696:Peptidylprolyl Isomerase / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012269:Ribosomal Proteins / Q000235:genetics; D012662:Semen Preservation; D012984:Software; D013094:Spermatozoa / Q000187:drug effects / Q000378:metabolism; D013552:Swine
D003451:Cryoprotective Agents; D012269:Ribosomal Proteins; C056368:ribosomal protein L4; D024581:Glyceraldehyde-3-Phosphate Dehydrogenase (Phosphorylating); D019696:Peptidylprolyl Isomerase
BestKeeper; Boar spermatozoa cryopreservation; NormFinder; Reference genes; geNorm