Expression stability of ACTB, 18S, and GAPDH in human placental tissues from subjects with PCOS and controls: GAPDH expression is increased in PCOS
Purpose The aim was to assess the expression stability of three commonly used reference genes, namely, β-actin ( ACTB ), 18S ribosomal RNA ( 18S ), and glyceraldehyde-3-phosphate dehydrogenase ( GAPDH ), in placental tissue obtained from pregnant women with polycystic ovary syndrome (PCOS) and healt...
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| Published in | Hormones (Athens, Greece) Vol. 21; no. 2; pp. 329 - 333 |
|---|---|
| Main Authors | , , , , , , |
| Format | Journal Article |
| Language | English |
| Published |
Cham
Springer International Publishing
01.06.2022
|
| Subjects | |
| Online Access | Get full text |
| ISSN | 1109-3099 2520-8721 2520-8721 |
| DOI | 10.1007/s42000-022-00372-z |
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| Abstract | Purpose
The aim was to assess the expression stability of three commonly used reference genes, namely, β-actin (
ACTB
), 18S ribosomal RNA (
18S
), and glyceraldehyde-3-phosphate dehydrogenase (
GAPDH
), in placental tissue obtained from pregnant women with polycystic ovary syndrome (PCOS) and healthy controls.
Methods
mRNA was isolated after delivery from the placentae of 10 PCOS and 10 control women with term, uncomplicated, singleton pregnancies. The expression of
ACTB
,
18S
, and
GAPDH
was analyzed using real-time polymerase chain reaction (RT-PCR). Gene expression stability was evaluated with the RefFinder, GeNorm, Normfinder, BestKeeper, and Delta-Ct tools.
Results
ACTB
was ranked as the most stably expressed gene, followed by
18S
. The expression of
GAPDH
varied considerably in both studied groups, while it was increased in PCOS versus controls (5.3-fold,
p
< 0.05).
Conclusions
ACTB
is an appropriate reference gene for placental gene expression studies in women with PCOS, whereas
GAPDH
is unfit for such a role, as its placental expression is increased in PCOS. |
|---|---|
| AbstractList | The aim was to assess the expression stability of three commonly used reference genes, namely, β-actin (ACTB), 18S ribosomal RNA (18S), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH), in placental tissue obtained from pregnant women with polycystic ovary syndrome (PCOS) and healthy controls.
mRNA was isolated after delivery from the placentae of 10 PCOS and 10 control women with term, uncomplicated, singleton pregnancies. The expression of ACTB, 18S, and GAPDH was analyzed using real-time polymerase chain reaction (RT-PCR). Gene expression stability was evaluated with the RefFinder, GeNorm, Normfinder, BestKeeper, and Delta-Ct tools.
ACTB was ranked as the most stably expressed gene, followed by 18S. The expression of GAPDH varied considerably in both studied groups, while it was increased in PCOS versus controls (5.3-fold, p < 0.05).
ACTB is an appropriate reference gene for placental gene expression studies in women with PCOS, whereas GAPDH is unfit for such a role, as its placental expression is increased in PCOS. The aim was to assess the expression stability of three commonly used reference genes, namely, β-actin (ACTB), 18S ribosomal RNA (18S), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH), in placental tissue obtained from pregnant women with polycystic ovary syndrome (PCOS) and healthy controls.PURPOSEThe aim was to assess the expression stability of three commonly used reference genes, namely, β-actin (ACTB), 18S ribosomal RNA (18S), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH), in placental tissue obtained from pregnant women with polycystic ovary syndrome (PCOS) and healthy controls.mRNA was isolated after delivery from the placentae of 10 PCOS and 10 control women with term, uncomplicated, singleton pregnancies. The expression of ACTB, 18S, and GAPDH was analyzed using real-time polymerase chain reaction (RT-PCR). Gene expression stability was evaluated with the RefFinder, GeNorm, Normfinder, BestKeeper, and Delta-Ct tools.METHODSmRNA was isolated after delivery from the placentae of 10 PCOS and 10 control women with term, uncomplicated, singleton pregnancies. The expression of ACTB, 18S, and GAPDH was analyzed using real-time polymerase chain reaction (RT-PCR). Gene expression stability was evaluated with the RefFinder, GeNorm, Normfinder, BestKeeper, and Delta-Ct tools.ACTB was ranked as the most stably expressed gene, followed by 18S. The expression of GAPDH varied considerably in both studied groups, while it was increased in PCOS versus controls (5.3-fold, p < 0.05).RESULTSACTB was ranked as the most stably expressed gene, followed by 18S. The expression of GAPDH varied considerably in both studied groups, while it was increased in PCOS versus controls (5.3-fold, p < 0.05).ACTB is an appropriate reference gene for placental gene expression studies in women with PCOS, whereas GAPDH is unfit for such a role, as its placental expression is increased in PCOS.CONCLUSIONSACTB is an appropriate reference gene for placental gene expression studies in women with PCOS, whereas GAPDH is unfit for such a role, as its placental expression is increased in PCOS. Purpose The aim was to assess the expression stability of three commonly used reference genes, namely, β-actin ( ACTB ), 18S ribosomal RNA ( 18S ), and glyceraldehyde-3-phosphate dehydrogenase ( GAPDH ), in placental tissue obtained from pregnant women with polycystic ovary syndrome (PCOS) and healthy controls. Methods mRNA was isolated after delivery from the placentae of 10 PCOS and 10 control women with term, uncomplicated, singleton pregnancies. The expression of ACTB , 18S , and GAPDH was analyzed using real-time polymerase chain reaction (RT-PCR). Gene expression stability was evaluated with the RefFinder, GeNorm, Normfinder, BestKeeper, and Delta-Ct tools. Results ACTB was ranked as the most stably expressed gene, followed by 18S . The expression of GAPDH varied considerably in both studied groups, while it was increased in PCOS versus controls (5.3-fold, p < 0.05). Conclusions ACTB is an appropriate reference gene for placental gene expression studies in women with PCOS, whereas GAPDH is unfit for such a role, as its placental expression is increased in PCOS. |
| Author | Panagodimou, Evangelia Koika, Vasiliki Kaponis, Apostolos Georgopoulos, Neoklis A. Markantes, Georgios K. Adonakis, George Markatos, Fotios |
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| Cites_doi | 10.1007/s12033-012-9539-2 10.1016/j.placenta.2016.10.017 10.1097/MD.0000000000004863 10.1023/B:BILE.0000019559.84305.47 10.1093/humupd/dmx013 10.1016/j.placenta.2008.06.007 10.1093/humrep/deu351 10.1093/humrep/det250 10.1016/j.ebiom.2020.102993 10.1186/gb-2002-3-7-research0034 10.1007/s00404-020-05495-5 10.1016/j.placenta.2004.09.009 10.1371/journal.pone.0122515 10.1158/0008-5472.CAN-04-0496 10.1186/1471-2199-7-33 10.2144/00292rv02 |
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| Keywords | Placenta Reference gene Gene expression RTPCR Polycystic ovary syndrome (PCOS) |
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| References | Yu HF, Chen HS, Rao DP, Gong J (2016) Association between polycystic ovary syndrome and the risk of pregnancy complications: a PRISMA-compliant systematic review and meta-analysis. Medicine (Baltimore) 95:e4863 MaliqueoMSundström PoromaaIVankyEFornesRBenrickAÅkerudHStridsklevSLabrieFJanssonTStener-VictorinEPlacental STAT3 signaling is activated in women with polycystic ovary syndromeHum Reprod2015306927001:CAS:528:DC%2BC2sXhslaiurvF10.1093/humrep/deu351 AndersenCLJensenJLOrntoftTFNormalization of real-time quantitative reverse transcription-PCR data: a model-based variance estimation approach to identify genes suited for normalization, applied to bladder and colon cancer data setsCancer Res200464524552501:CAS:528:DC%2BD2cXmtF2mtbg%3D10.1158/0008-5472.CAN-04-0496 De Spiegelaere W, Dern-Wieloch J, Weigel R, Schumacher V, Schorle H, Nettersheim D, Bergmann M, Brehm R, Kliesch S, Vandekerckhove L, Fink C (2015) Reference gene validation for RT-qPCR, a note on different available software packages. PLoS One 10:e0122515 Wang F, Xie N, Zhou J, Dai M, Zhang Q, Hardiman PJ, Qu F (2020) Molecular mechanisms underlying altered neurobehavioural development of female offspring of mothers with polycystic ovary syndrome: FOS-mediated regulation of neurotrophins in placenta. EBioMedicine 60:102993 LanoixDLacasseAASt-PierreJTaylorSCEthier-ChiassonMLafondJVaillancourtCQuantitative PCR pitfalls: the case of the human placentaMol Biotechnol2012522342431:CAS:528:DC%2BC38XhsFSqsL3J10.1007/s12033-012-9539-2 MellerMVadachkoriaSLuthyDAWilliamsMAEvaluation of housekeeping genes in placental comparative expression studiesPlacenta2005266016071:CAS:528:DC%2BD2MXns1WgsLk%3D10.1016/j.placenta.2004.09.009 PfafflMWTichopadAPrgometCNeuviansTPDetermination of stable housekeeping genes, differentially regulated target genes and sample integrity: BestKeeper–Excel-based tool using pair-wise correlationsBiotechol Lett2004265095151:CAS:528:DC%2BD2cXhvF2mtLk%3D10.1023/B:BILE.0000019559.84305.47 FilippouPHomburgRIs foetal hyperexposure to androgens a cause of PCOS?Hum Reprod Update2017234214321:CAS:528:DC%2BC1MXjsFCit78%3D10.1093/humupd/dmx013 ZhaoCZhouYShenXGongMLuYFangCChenJJuRCircular RNA expression profiling in the fetal side of placenta from maternal polycystic ovary syndrome and circ_0023942 inhibits the proliferation of human ovarian granulosa cellArch Gynecol Obstet20203019639711:CAS:528:DC%2BB3cXlslGgtLo%3D10.1007/s00404-020-05495-5 Vandesompele J, De Preter K, Pattyn F, Poppe B, Van Roy N, De Paepe A, Speleman F (2002) Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes. Genome Biol 3:RESEARCH0034 SuzukiTHigginsPJCrawfordDRControl selection for RNA quantitationBiotechniques2000293323371:CAS:528:DC%2BD3cXls12jsr0%3D10.2144/00292rv02 MurthiPFitzpatrickEBorgAJDonathSBrenneckeSPKalionisBGAPDH, 18S rRNA and YWHAZ are suitable endogenous reference genes for relative gene expression studies in placental tissues from human idiopathic fetal growth restrictionPlacenta2008297988011:CAS:528:DC%2BD1cXhtVOhsrvF10.1016/j.placenta.2008.06.007 LiYLuHJiYWuSYangYIdentification of genes for normalization of real-time RT-PCR data in placental tissues from intrahepatic cholestasis of pregnancyPlacenta2016481331351:CAS:528:DC%2BC28XhvVWmtbbP10.1016/j.placenta.2016.10.017 PalombaSRussoTFalboADi CelloATolinoATucciLLa SalaGBZulloFMacroscopic and microscopic findings of the placenta in women with polycystic ovary syndromeHum Reprod201328283828471:STN:280:DC%2BC3sjht1KlsQ%3D%3D10.1093/humrep/det250 SilverNBestSJiangJTheinSLSelection of housekeeping genes for gene expression studies in human reticulocytes using real-time PCRBMC Mol Biol200673310.1186/1471-2199-7-33 MW Pfaffl (372_CR9) 2004; 26 D Lanoix (372_CR11) 2012; 52 S Palomba (372_CR16) 2013; 28 P Murthi (372_CR5) 2008; 29 T Suzuki (372_CR14) 2000; 29 Y Li (372_CR12) 2016; 48 M Maliqueo (372_CR3) 2015; 30 372_CR7 372_CR1 N Silver (372_CR10) 2006; 7 C Zhao (372_CR6) 2020; 301 M Meller (372_CR4) 2005; 26 CL Andersen (372_CR8) 2004; 64 372_CR15 P Filippou (372_CR2) 2017; 23 372_CR13 |
| References_xml | – reference: Wang F, Xie N, Zhou J, Dai M, Zhang Q, Hardiman PJ, Qu F (2020) Molecular mechanisms underlying altered neurobehavioural development of female offspring of mothers with polycystic ovary syndrome: FOS-mediated regulation of neurotrophins in placenta. EBioMedicine 60:102993 – reference: Yu HF, Chen HS, Rao DP, Gong J (2016) Association between polycystic ovary syndrome and the risk of pregnancy complications: a PRISMA-compliant systematic review and meta-analysis. Medicine (Baltimore) 95:e4863 – reference: LiYLuHJiYWuSYangYIdentification of genes for normalization of real-time RT-PCR data in placental tissues from intrahepatic cholestasis of pregnancyPlacenta2016481331351:CAS:528:DC%2BC28XhvVWmtbbP10.1016/j.placenta.2016.10.017 – reference: AndersenCLJensenJLOrntoftTFNormalization of real-time quantitative reverse transcription-PCR data: a model-based variance estimation approach to identify genes suited for normalization, applied to bladder and colon cancer data setsCancer Res200464524552501:CAS:528:DC%2BD2cXmtF2mtbg%3D10.1158/0008-5472.CAN-04-0496 – reference: PfafflMWTichopadAPrgometCNeuviansTPDetermination of stable housekeeping genes, differentially regulated target genes and sample integrity: BestKeeper–Excel-based tool using pair-wise correlationsBiotechol Lett2004265095151:CAS:528:DC%2BD2cXhvF2mtLk%3D10.1023/B:BILE.0000019559.84305.47 – reference: FilippouPHomburgRIs foetal hyperexposure to androgens a cause of PCOS?Hum Reprod Update2017234214321:CAS:528:DC%2BC1MXjsFCit78%3D10.1093/humupd/dmx013 – reference: ZhaoCZhouYShenXGongMLuYFangCChenJJuRCircular RNA expression profiling in the fetal side of placenta from maternal polycystic ovary syndrome and circ_0023942 inhibits the proliferation of human ovarian granulosa cellArch Gynecol Obstet20203019639711:CAS:528:DC%2BB3cXlslGgtLo%3D10.1007/s00404-020-05495-5 – reference: PalombaSRussoTFalboADi CelloATolinoATucciLLa SalaGBZulloFMacroscopic and microscopic findings of the placenta in women with polycystic ovary syndromeHum Reprod201328283828471:STN:280:DC%2BC3sjht1KlsQ%3D%3D10.1093/humrep/det250 – reference: Vandesompele J, De Preter K, Pattyn F, Poppe B, Van Roy N, De Paepe A, Speleman F (2002) Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes. Genome Biol 3:RESEARCH0034 – reference: MurthiPFitzpatrickEBorgAJDonathSBrenneckeSPKalionisBGAPDH, 18S rRNA and YWHAZ are suitable endogenous reference genes for relative gene expression studies in placental tissues from human idiopathic fetal growth restrictionPlacenta2008297988011:CAS:528:DC%2BD1cXhtVOhsrvF10.1016/j.placenta.2008.06.007 – reference: MaliqueoMSundström PoromaaIVankyEFornesRBenrickAÅkerudHStridsklevSLabrieFJanssonTStener-VictorinEPlacental STAT3 signaling is activated in women with polycystic ovary syndromeHum Reprod2015306927001:CAS:528:DC%2BC2sXhslaiurvF10.1093/humrep/deu351 – reference: SilverNBestSJiangJTheinSLSelection of housekeeping genes for gene expression studies in human reticulocytes using real-time PCRBMC Mol Biol200673310.1186/1471-2199-7-33 – reference: LanoixDLacasseAASt-PierreJTaylorSCEthier-ChiassonMLafondJVaillancourtCQuantitative PCR pitfalls: the case of the human placentaMol Biotechnol2012522342431:CAS:528:DC%2BC38XhsFSqsL3J10.1007/s12033-012-9539-2 – reference: De Spiegelaere W, Dern-Wieloch J, Weigel R, Schumacher V, Schorle H, Nettersheim D, Bergmann M, Brehm R, Kliesch S, Vandekerckhove L, Fink C (2015) Reference gene validation for RT-qPCR, a note on different available software packages. PLoS One 10:e0122515 – reference: SuzukiTHigginsPJCrawfordDRControl selection for RNA quantitationBiotechniques2000293323371:CAS:528:DC%2BD3cXls12jsr0%3D10.2144/00292rv02 – reference: MellerMVadachkoriaSLuthyDAWilliamsMAEvaluation of housekeeping genes in placental comparative expression studiesPlacenta2005266016071:CAS:528:DC%2BD2MXns1WgsLk%3D10.1016/j.placenta.2004.09.009 – volume: 52 start-page: 234 year: 2012 ident: 372_CR11 publication-title: Mol Biotechnol doi: 10.1007/s12033-012-9539-2 – volume: 48 start-page: 133 year: 2016 ident: 372_CR12 publication-title: Placenta doi: 10.1016/j.placenta.2016.10.017 – ident: 372_CR1 doi: 10.1097/MD.0000000000004863 – volume: 26 start-page: 509 year: 2004 ident: 372_CR9 publication-title: Biotechol Lett doi: 10.1023/B:BILE.0000019559.84305.47 – volume: 23 start-page: 421 year: 2017 ident: 372_CR2 publication-title: Hum Reprod Update doi: 10.1093/humupd/dmx013 – volume: 29 start-page: 798 year: 2008 ident: 372_CR5 publication-title: Placenta doi: 10.1016/j.placenta.2008.06.007 – volume: 30 start-page: 692 year: 2015 ident: 372_CR3 publication-title: Hum Reprod doi: 10.1093/humrep/deu351 – volume: 28 start-page: 2838 year: 2013 ident: 372_CR16 publication-title: Hum Reprod doi: 10.1093/humrep/det250 – ident: 372_CR15 doi: 10.1016/j.ebiom.2020.102993 – ident: 372_CR7 doi: 10.1186/gb-2002-3-7-research0034 – volume: 301 start-page: 963 year: 2020 ident: 372_CR6 publication-title: Arch Gynecol Obstet doi: 10.1007/s00404-020-05495-5 – volume: 26 start-page: 601 year: 2005 ident: 372_CR4 publication-title: Placenta doi: 10.1016/j.placenta.2004.09.009 – ident: 372_CR13 doi: 10.1371/journal.pone.0122515 – volume: 64 start-page: 5245 year: 2004 ident: 372_CR8 publication-title: Cancer Res doi: 10.1158/0008-5472.CAN-04-0496 – volume: 7 start-page: 33 year: 2006 ident: 372_CR10 publication-title: BMC Mol Biol doi: 10.1186/1471-2199-7-33 – volume: 29 start-page: 332 year: 2000 ident: 372_CR14 publication-title: Biotechniques doi: 10.2144/00292rv02 |
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The aim was to assess the expression stability of three commonly used reference genes, namely, β-actin (
ACTB
), 18S ribosomal RNA (
18S
), and... The aim was to assess the expression stability of three commonly used reference genes, namely, β-actin (ACTB), 18S ribosomal RNA (18S), and... |
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| Title | Expression stability of ACTB, 18S, and GAPDH in human placental tissues from subjects with PCOS and controls: GAPDH expression is increased in PCOS |
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