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Measurement of Housekeeping Genes in Human Placenta
Placenta 30 (2009) 1002–1003
Contents lists available at ScienceDirect
Placenta journal homepage: www.elsevier.com/locate/placenta
Letter to the Editor
Measurement of Housekeeping Genes in Human Placenta
The measurement of mRNA expression in the placenta is an important tool for the investigation of placental function and the identification of markers that can indicate placental or fetal health. The comparison of mRNA expression by real-time RT-PCR requires normalization by appropriate endogenous housekeeping genes which are constitutively expressed in the tissue and do not vary due to the experimental conditions. This controls for variables such as the amount of starting material, enzymatic efficiencies, and differences between tissues or cells in overall transcriptional activity [1]. Identifying stable control genes in placental tissue is important to ensure the validity of gene expression studies, as normalizing the gene of interest using the geometric mean [1] of the control genes increases the quality of the data. The Genorm house keeping selection kit provides a way to assess the variability of either 6 or 12 commonly used control genes in a tissue (www.primerdesign.co.uk). Two previous papers have reported Genorm analysis in placental tissue, on 6 and 7 genes, 5 of which were the same [2,3]. Our group has added to this work by analyzing the stability of 12 housekeeping genes in placental RNA extracted by two different methods. Placentas were collected from normal term pregnancies immediately after delivery, following written informed consent and with the approval of the South and West Hants Local Research Ethics Committee. Five villous tissue samples were selected from each placenta using a stratified random sampling method, snap frozen in liquid nitrogen, and stored at 80 C. To ensure that the RNA extracted was representative of the placenta as a whole the five villous tissue samples were pooled and powdered. For 10 placentas total RNA was extracted from 30 mg powdered tissue using the RNeasy Fibrous Mini Kit (Qiagen Inc.) standard protocol with a DNase step. In a second group of 10 placentas total RNA was extracted from 30 mg powdered tissue using the trizol method. These two methods represent affinity column based RNA extraction and RNA extraction using organic solvents. Total RNA was reverse transcribed to produce cDNA. The mRNA expression of adenosine triphosphate synthase (ATP5B), beta actin (b-ACT), beta-2-microglobulin (b2 M), cytochrome c-1 (CYC1), eukaryotic translation initiation factor 4A isoform 2 (EIF4A2), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), succinate dehydrogenase complex (SDHA), splicing factor 3a subunit 1 (SF3A1), topoisomerase DNA I (TOP1), ubiquitin C (UBC), phospholipase A2 (YWHAZ) and 18s rRNA were measured by real-time PCR using primers and perfect probe (primer design). All samples were measured in duplicate using an ABI PRISM 7700 sequence detector system (Applied Bio-systems, California, USA). The cycle parameters were 95 C for 10 min, followed by 50 cycles of 95 C for 15 s, 50 C for 30 s and 72 C for 15 s. Cycle threshold (CT) values were
0143-4004/$ – see front matter Ó 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.placenta.2009.09.002
transformed into relative quantification data using the deltaCT method and expression stability and the optimum number of control genes required was calculated using geNorm software. As previous studies have shown [4], 18s rRNA had extremely high expression levels in the human placenta: indicated by a low CT value of 9.02 0.3 compared to a CT value ranging from 21.41 0.3 to 28.40 0.3 in the other genes. 18s rRNA expression is considerably higher than the majority of genes and we did not feel that it was directly comparable, so the analysis was performed without including this gene. The eleven other genes were ordered according to stability (Fig.1a) and the number of genes required for normalization determined (Fig. 1b). The normalization factor used is the geometric mean of the most stable housekeeping genes. To establish the number of genes used to make this geometric mean Vandesompele et al. 2002 devised a calculation whereby a value called the V score is determined. The V score indicates how the normalization factor changes when another gene is included in its calculation. They established that a V score of 0.15 or below indicated that the additional gene had no significant contribution to the newly calculated normalization factor and was therefore not needed. For both the RNeasy kit and trizol data, the pair-wise variation with the sequential addition of each housekeeping gene indicated that two genes could potentially be used as the addition of a third gene gave a V score below 0.15 (Fig. 1b). The consensus of opinion is however the use of a third gene for increased accuracy of normalization [1–3]. In the samples with mRNA extracted using the trizol method UBC and TOP1 showed the highest stability followed by EIF4AT and then YWHAZ (Fig. 1a). In the samples with mRNA extracted using the RNeasy kit CYC1 and UBC showed the highest stability followed by YWHAZ and then TOP1 (Fig. 1a). The results obtained were broadly similar, although there were differences in the gene stability ranking between the two RNA extraction methods. The identification of YWHAZ as a stable housekeeping gene is consistent with the studies of both Mellor et al., 2005 [2] and Murthi et al., 2008 [3] which used placentas from diabetic and growth restricted pregnancies respectively. While our study and those of Mellor and Murthi, found that the commonly used housekeeping genes GAPDH and b-actin were relatively stable, our studies suggests that other genes are much more stably expressed in placenta. Previous studies have also suggested that the expression of GAPDH and b-actin in placenta is affected by a number of conditions such as hypoxia and gestational age [4,5]. Our study of 12 genes found that genes not previously tested in placenta were more stable than those identified as most stable by these previous studies [2,3]. We therefore suggest UBC, TOP1 and YWHAZ as possible genes recommended for
Letter to the Editor / Placenta 30 (2009) 1002–1003
1003
a
b
Fig. 1. a) The most stably expressed housekeeping genes. A graph showing the average expression stability value (M) for each housekeeping gene ranked according to increasing stability with the most stable genes on the right (geNorm 3.4). b) The number of genes required for normalization. A graph showing the levels of variation in average housekeeping gene stability with the sequential addition of each housekeeping gene to the equation (for calculating the normalization factor). Starting with the most stably expressed genes on the left with the inclusion of a 3rd, 4th, 5th gene etc. moving to the right (geNorm 3.4). The graph indicates that the two most stable genes create a normalization factor which is not significantly altered by the addition of the other genes as they all have a V score below 0.15.
normalization, as these appear to be the most stably expressed genes regardless of extraction procedure. While the genes we identify are applicable to studies of normal placenta the investigation of a wider range of control genes may be appropriate in order to identify the best candidates for studies of pathological pregnancies. Acknowledgements PD was supported by the Gerald Kercut Trust References [1] Vandesompele J, De PK, Pattyn F, Poppe B, Van RN, De PA, et al. Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes. Genome Biol 2002;3. RESEARCH0034. [2] Meller M, Vadachkoria S, Luthy DA, Williams MA. Evaluation of housekeeping genes in placental comparative expression studies. Placenta 2005;26:601–7.
[3] Murthi P, Fitzpatrick E, Borg AJ, Donath S, Brennecke SP, Kalionis BGAPDH. 18S rRNA and YWHAZ are suitable endogenous reference genes for relative gene expression studies in placental tissues from human idiopathic fetal growth restriction. Placenta 2008;29:798–801. [4] Patel P, Boyd CA, Johnston DG, Williamson C. Analysis of GAPDH as a standard for gene expression quantification in human placenta. Placenta 2002;23:697–8. [5] Suzuki T, Higgins PJ, Crawford DR. Control selection for RNA quantitation. Biotechniques 2000;29:332–7.
J.K. Cleal*, P. Day, M.A. Hanson, R.M. Lewis University of southampton, Institute of Developmental Sciences, Mail point 887, Southampton General Hospital, Tremona Road, Southampton SO166YD United Kingdom * Corresponding author. Tel.: þ44 2380798663. E-mail address: [email protected] (J.K. Cleal) Accepted 14 September 2009
Contents lists available at ScienceDirect
Placenta journal homepage: www.elsevier.com/locate/placenta
Letter to the Editor
Measurement of Housekeeping Genes in Human Placenta
The measurement of mRNA expression in the placenta is an important tool for the investigation of placental function and the identification of markers that can indicate placental or fetal health. The comparison of mRNA expression by real-time RT-PCR requires normalization by appropriate endogenous housekeeping genes which are constitutively expressed in the tissue and do not vary due to the experimental conditions. This controls for variables such as the amount of starting material, enzymatic efficiencies, and differences between tissues or cells in overall transcriptional activity [1]. Identifying stable control genes in placental tissue is important to ensure the validity of gene expression studies, as normalizing the gene of interest using the geometric mean [1] of the control genes increases the quality of the data. The Genorm house keeping selection kit provides a way to assess the variability of either 6 or 12 commonly used control genes in a tissue (www.primerdesign.co.uk). Two previous papers have reported Genorm analysis in placental tissue, on 6 and 7 genes, 5 of which were the same [2,3]. Our group has added to this work by analyzing the stability of 12 housekeeping genes in placental RNA extracted by two different methods. Placentas were collected from normal term pregnancies immediately after delivery, following written informed consent and with the approval of the South and West Hants Local Research Ethics Committee. Five villous tissue samples were selected from each placenta using a stratified random sampling method, snap frozen in liquid nitrogen, and stored at 80 C. To ensure that the RNA extracted was representative of the placenta as a whole the five villous tissue samples were pooled and powdered. For 10 placentas total RNA was extracted from 30 mg powdered tissue using the RNeasy Fibrous Mini Kit (Qiagen Inc.) standard protocol with a DNase step. In a second group of 10 placentas total RNA was extracted from 30 mg powdered tissue using the trizol method. These two methods represent affinity column based RNA extraction and RNA extraction using organic solvents. Total RNA was reverse transcribed to produce cDNA. The mRNA expression of adenosine triphosphate synthase (ATP5B), beta actin (b-ACT), beta-2-microglobulin (b2 M), cytochrome c-1 (CYC1), eukaryotic translation initiation factor 4A isoform 2 (EIF4A2), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), succinate dehydrogenase complex (SDHA), splicing factor 3a subunit 1 (SF3A1), topoisomerase DNA I (TOP1), ubiquitin C (UBC), phospholipase A2 (YWHAZ) and 18s rRNA were measured by real-time PCR using primers and perfect probe (primer design). All samples were measured in duplicate using an ABI PRISM 7700 sequence detector system (Applied Bio-systems, California, USA). The cycle parameters were 95 C for 10 min, followed by 50 cycles of 95 C for 15 s, 50 C for 30 s and 72 C for 15 s. Cycle threshold (CT) values were
0143-4004/$ – see front matter Ó 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.placenta.2009.09.002
transformed into relative quantification data using the deltaCT method and expression stability and the optimum number of control genes required was calculated using geNorm software. As previous studies have shown [4], 18s rRNA had extremely high expression levels in the human placenta: indicated by a low CT value of 9.02 0.3 compared to a CT value ranging from 21.41 0.3 to 28.40 0.3 in the other genes. 18s rRNA expression is considerably higher than the majority of genes and we did not feel that it was directly comparable, so the analysis was performed without including this gene. The eleven other genes were ordered according to stability (Fig.1a) and the number of genes required for normalization determined (Fig. 1b). The normalization factor used is the geometric mean of the most stable housekeeping genes. To establish the number of genes used to make this geometric mean Vandesompele et al. 2002 devised a calculation whereby a value called the V score is determined. The V score indicates how the normalization factor changes when another gene is included in its calculation. They established that a V score of 0.15 or below indicated that the additional gene had no significant contribution to the newly calculated normalization factor and was therefore not needed. For both the RNeasy kit and trizol data, the pair-wise variation with the sequential addition of each housekeeping gene indicated that two genes could potentially be used as the addition of a third gene gave a V score below 0.15 (Fig. 1b). The consensus of opinion is however the use of a third gene for increased accuracy of normalization [1–3]. In the samples with mRNA extracted using the trizol method UBC and TOP1 showed the highest stability followed by EIF4AT and then YWHAZ (Fig. 1a). In the samples with mRNA extracted using the RNeasy kit CYC1 and UBC showed the highest stability followed by YWHAZ and then TOP1 (Fig. 1a). The results obtained were broadly similar, although there were differences in the gene stability ranking between the two RNA extraction methods. The identification of YWHAZ as a stable housekeeping gene is consistent with the studies of both Mellor et al., 2005 [2] and Murthi et al., 2008 [3] which used placentas from diabetic and growth restricted pregnancies respectively. While our study and those of Mellor and Murthi, found that the commonly used housekeeping genes GAPDH and b-actin were relatively stable, our studies suggests that other genes are much more stably expressed in placenta. Previous studies have also suggested that the expression of GAPDH and b-actin in placenta is affected by a number of conditions such as hypoxia and gestational age [4,5]. Our study of 12 genes found that genes not previously tested in placenta were more stable than those identified as most stable by these previous studies [2,3]. We therefore suggest UBC, TOP1 and YWHAZ as possible genes recommended for
Letter to the Editor / Placenta 30 (2009) 1002–1003
1003
a
b
Fig. 1. a) The most stably expressed housekeeping genes. A graph showing the average expression stability value (M) for each housekeeping gene ranked according to increasing stability with the most stable genes on the right (geNorm 3.4). b) The number of genes required for normalization. A graph showing the levels of variation in average housekeeping gene stability with the sequential addition of each housekeeping gene to the equation (for calculating the normalization factor). Starting with the most stably expressed genes on the left with the inclusion of a 3rd, 4th, 5th gene etc. moving to the right (geNorm 3.4). The graph indicates that the two most stable genes create a normalization factor which is not significantly altered by the addition of the other genes as they all have a V score below 0.15.
normalization, as these appear to be the most stably expressed genes regardless of extraction procedure. While the genes we identify are applicable to studies of normal placenta the investigation of a wider range of control genes may be appropriate in order to identify the best candidates for studies of pathological pregnancies. Acknowledgements PD was supported by the Gerald Kercut Trust References [1] Vandesompele J, De PK, Pattyn F, Poppe B, Van RN, De PA, et al. Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes. Genome Biol 2002;3. RESEARCH0034. [2] Meller M, Vadachkoria S, Luthy DA, Williams MA. Evaluation of housekeeping genes in placental comparative expression studies. Placenta 2005;26:601–7.
[3] Murthi P, Fitzpatrick E, Borg AJ, Donath S, Brennecke SP, Kalionis BGAPDH. 18S rRNA and YWHAZ are suitable endogenous reference genes for relative gene expression studies in placental tissues from human idiopathic fetal growth restriction. Placenta 2008;29:798–801. [4] Patel P, Boyd CA, Johnston DG, Williamson C. Analysis of GAPDH as a standard for gene expression quantification in human placenta. Placenta 2002;23:697–8. [5] Suzuki T, Higgins PJ, Crawford DR. Control selection for RNA quantitation. Biotechniques 2000;29:332–7.
J.K. Cleal*, P. Day, M.A. Hanson, R.M. Lewis University of southampton, Institute of Developmental Sciences, Mail point 887, Southampton General Hospital, Tremona Road, Southampton SO166YD United Kingdom * Corresponding author. Tel.: þ44 2380798663. E-mail address: [email protected] (J.K. Cleal) Accepted 14 September 2009