Skip to main content
SpringerLink
Log in

Quantitative PCR Pitfalls: The Case of the Human Placenta

  • Research
  • Published:
Molecular Biotechnology Aims and scope Submit manuscript

An Erratum to this article was published on 12 June 2012

Abstract

Reverse-transcription quantitative polymerase chain reaction (RT-qPCR) is a rapid and high throughput gene expression quantification technology. In order to obtain accurate results, several key experimental design and standardization steps must be rigorously followed as previously described in the Minimum Information for Publication of Quantitative Real-Time PCR Experiments (MIQE) guidelines. This study investigates the effect of reference gene normalization and the impact of RNA degradation on gene expression of 8-oxoguanine DNA glycosylase in human placenta from pregnancies complicated by preeclampsia and gestational diabetes mellitus and their gestation-matched controls. The data presented here show how RNA quality and appropriate reference gene selection is not only important to obtain accurate and reproducible RT-qPCR data but how different and even opposite results can be reported if the key steps outlined in the MIQE guidelines are not followed. The procedures and associated results presented in this study provide the first practical application of the MIQE guidelines to placental analysis in normal and pathological pregnancies.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5

Similar content being viewed by others

References

  1. Bustin, S. A., Benes, V., Garson, J. A., Hellemans, J., Huggett, J., Kubista, M., et al. (2009). The MIQE guidelines: minimum information for publication of quantitative real-time PCR experiments. Clinical Chemistry, 55(4), 611–622.

    Article  CAS  Google Scholar 

  2. Bustin, S. A. (2010). Why the need for qPCR publication guidelines? The case for MIQE. Methods, 50(4), 217–226.

    Article  CAS  Google Scholar 

  3. Bustin, S. A., Vandesompele, J., & Pfaffl, M. W. (2009). Standardization of qPCR and RT-qPCR. Genetic Engineering & Biotechnology News, 29, 14.

    Google Scholar 

  4. Bohlenius, H., Eriksson, S., Parcy, F., & Nilsson, O. (2007). Retraction. Science, 316(5823), 367.

    Article  CAS  Google Scholar 

  5. The Editors of Lancet. (2010). Retraction—Ileal-lymphoid-nodular hyperplasia, non-specific colitis, and pervasive developmental disorder in children. Lancet, 375(9713), 445.

    Article  Google Scholar 

  6. Vermeulen, J., De Preter, K., Lefever, S., et al. (2011). Measurable impact of RNA quality on gene expression results from quantitative PCR. Nucleic Acids Research, 39, e63.

    Article  CAS  Google Scholar 

  7. Holland, N. T., Smith, M. T., Eskenazi, B., & Bastaki, M. (2003). Biological sample collection and processing for molecular epidemiological studies. Mutation Research, 543(3), 217–234.

    Article  CAS  Google Scholar 

  8. Schoor, O., Weinschenk, T., Hennenlotter. J., Corvin, S., Stenzl, A., Rammensee, H. G., Stevanovic, S. (2003). Moderate degradation does not preclude microarray analysis of small amounts of RNA. Biotechniques 35(6), 1192–6, 1198–1201.

    Google Scholar 

  9. Budhraja, V., & Sadovsky, Y. (2006). Microarray analysis of trophoblast cells. Methods in Molecular Medicine, 121, 411–424.

    CAS  Google Scholar 

  10. Meller, M., Vadachkoria, S., Luthy, D. A., & Williams, M. A. (2005). Evaluation of housekeeping genes in placental comparative expression studies. Placenta, 26(8–9), 601–607.

    Article  CAS  Google Scholar 

  11. Murthi, P., Fitzpatrick, E., Borg, A. J., Donath, S., Brennecke, S. P., & Kalionis, B. (2008). GAPDH, 18S rRNA and YWHAZ are suitable endogenous reference genes for relative gene expression studies in placental tissues from human idiopathic fetal growth restriction. Placenta, 29(9), 798–801.

    Article  CAS  Google Scholar 

  12. Cleal, J. K., Day, P., Hanson, M. A., & Lewis, R. M. (2009). Measurement of housekeeping genes in human placenta. Placenta, 30(11), 1002–1003.

    Article  CAS  Google Scholar 

  13. Murphy, C. L., & Polak, J. M. (2002). Differentiating embryonic stem cells: GAPDH, but neither HPRT nor beta-tubulin is suitable as an internal standard for measuring RNA levels. Tissue Engineering, 8(4), 551–559.

    Article  CAS  Google Scholar 

  14. Steele, B. K., Meyers, C., & Ozbun, M. A. (2002). Variable expression of some “housekeeping” genes during human keratinocyte differentiation. Analytical Biochemistry, 307(2), 341–347.

    Article  CAS  Google Scholar 

  15. Hamalainen, H. K., Tubman, J. C., Vikman, S., Kyrola, T., Ylikoski, E., Warrington, J. A., et al. (2001). Identification and validation of endogenous reference genes for expression profiling of T helper cell differentiation by quantitative real-time RT-PCR. Analytical Biochemistry, 299(1), 63–70.

    Article  CAS  Google Scholar 

  16. Yamada, H., Chen, D., Monstein, H. J., & Hakanson, R. (1997). Effects of fasting on the expression of gastrin, cholecystokinin, and somatostatin genes and of various housekeeping genes in the pancreas and upper digestive tract of rats. Biochemical and Biophysical Research Communications, 231(3), 835–838.

    Article  CAS  Google Scholar 

  17. Coughlan, M. T., Vervaart, P. P., Permezel, M., Georgiou, H. M., & Rice, G. E. (2004). Altered placental oxidative stress status in gestational diabetes mellitus. Placenta, 25(1), 78–84.

    Article  CAS  Google Scholar 

  18. Redman, C. W., & Sargent, I. L. (2009). Placental stress and pre-eclampsia: A revised view. Placenta. 30(Suppl A), S38–S42.

    Google Scholar 

  19. Huppertz, B. (2008). Placental origins of preeclampsia: challenging the current hypothesis. Hypertension, 51(4), 970–975.

    Article  CAS  Google Scholar 

  20. Jawerbaum, A., Capobianco, E., Pustovrh, C., White, V., Baier, M., Salzberg, S., et al. (2004). Influence of peroxisome proliferator-activated receptor gamma activation by its endogenous ligand 15-deoxy Delta12,14 prostaglandin J2 on nitric oxide production in term placental tissues from diabetic women. Molecular Human Reproduction, 10(9), 671–676.

    Article  CAS  Google Scholar 

  21. Andersen, C. L., Jensen, J. L., & Orntoft, T. F. (2004). Normalization 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 sets. Cancer Research, 64(15), 5245–5250.

    Article  CAS  Google Scholar 

  22. 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 Biology 23(7), RESEARCH0034.

    Google Scholar 

  23. Pfaffl, M. W., Tichopad, A., Prgomet, C., & Neuvians, T. P. (2004). Determination of stable housekeeping genes, differentially regulated target genes and sample integrity: BestKeeper-Excel-based tool using pair-wise correlations. Biotechnology Letters, 26(6), 509–515.

    Article  CAS  Google Scholar 

  24. Jin, Z. G., Melaragno, M. G., Liao, D. F., Yan, C., Haendeler, J., Suh, Y. A., et al. (2000). Cyclophilin A is a secreted growth factor induced by oxidative stress. Circulation Research, 87(9), 789–796.

    Article  CAS  Google Scholar 

  25. American Diabetes Association. (2007). Standards of medical care in diabetes—2007. Diabetes Care, 30(Suppl 1), S4–S41.

    Article  Google Scholar 

  26. Mayhew, T. M. (2008). Taking tissue samples from the placenta: an illustration of principles and strategies. Placenta, 29(1), 1–14.

    Article  CAS  Google Scholar 

  27. Zuker, M. (2003). Mfold web server for nucleic acid folding and hybridization prediction. Nucleic Acids Research, 31(13), 3406–3415.

    Article  CAS  Google Scholar 

  28. Copois, V., Bibeau, F., Bascoul-Mollevi, C., Salvetat, N., Chalbos, P., Bareil, C., et al. (2007). Impact of RNA degradation on gene expression profiles: Assessment of different methods to reliably determine RNA quality. Journal of Biotechnology, 127(4), 549–559.

    Article  CAS  Google Scholar 

  29. Jahn, C. E., Charkowski, A. O., & Willis, D. K. (2008). Evaluation of isolation methods and RNA integrity for bacterial RNA quantitation. Journal of Microbiol Methods, 75(2), 318–324.

    Article  CAS  Google Scholar 

  30. Denisov V, Strong W, Walder M, Gingrich J, & Wintz H. (2008). Development and validation of RQI: An RNA quality indicator for Experion ™ automated electrophoresis system. BioRad Bulletin #5761.

  31. Hache, S., Takser, L., Lebellego, F., Weiler, H., Leduc, L., Forest, J. C., et al. (2011). Alteration of calcium homeostasis in primary preeclamptic syncytiotrophoblasts: effect on calcium exchange in placenta. Journal of Cellular and Molecular Medicine, 15(3), 654–667.

    Article  CAS  Google Scholar 

  32. Taylor, S., Wakem, M., Dijkman, G., Alsarraj, M., & Nguyen, M. (2010). A practical approach to RT-qPCR-Publishing data that conform to the MIQE guidelines. Methods, 50(4), S1–S5.

    Article  CAS  Google Scholar 

  33. Lanoix, D., St-Pierre, J., Lacasse, AA., Viau, M., Lafond, J., & Vaillancourt, C. (2012). Stability of reference proteins in human placenta: General protein stains are the benchmark. Placenta, 33(3), 151–156.

    Google Scholar 

  34. Patel, P., Boyd, C. A., Johnston, D. G., & Williamson, C. (2002). Analysis of GAPDH as a standard for gene expression quantification in human placenta. Placenta, 23(8–9), 697–698.

    Article  CAS  Google Scholar 

Download references

Acknowledgments

This study was financially supported by grants from the Natural Sciences and Engineering Research Council of Canada (NSERC # 262011-2009) awarded to C Vaillancourt and from the Canadian Institute of Health Research (CIHR NET 2002-2007) awarded to J Lafond. D Lanoix received studentships from the Fond de la recherche en Santé du Québec (FRSQ), AA Lacasse from the Fonds québécois de la recherche sur la nature et les technologies (FQRNT) and the NSERC and J St-Pierre from the Réseau Québécois en reproduction (RQR)-NSERC-CREATE scholarship program.

Author information

Authors and Affiliations

  1. INRS-Institut Armand-Frappier, Université du Québec, 531, boul. des Prairies, Building 18, Laval, QC, H7V 1B7, Canada

    Dave Lanoix, Andrée-Anne Lacasse, Joey St-Pierre & Cathy Vaillancourt

  2. BioMed Research Center, Montreal, QC, H3C 3P8, Canada

    Dave Lanoix, Andrée-Anne Lacasse, Joey St-Pierre, Maude Ethier-Chiasson, Julie Lafond & Cathy Vaillancourt

  3. Bio-Rad Laboratories (Canada) Ltd, 1329 Meyerside Drive, Mississauga, ON, L4C 1N9, Canada

    Sean C. Taylor

  4. Laboratoire de Physiologie materno-fœtale, Department of Biological Sciences, BioMed Research Center, Montreal, QC, H3C 3P8, Canada

    Maude Ethier-Chiasson & Julie Lafond

Authors
  1. Dave Lanoix
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Andrée-Anne Lacasse
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Joey St-Pierre
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Sean C. Taylor
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Maude Ethier-Chiasson
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Julie Lafond
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Cathy Vaillancourt
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Cathy Vaillancourt.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Lanoix, D., Lacasse, AA., St-Pierre, J. et al. Quantitative PCR Pitfalls: The Case of the Human Placenta. Mol Biotechnol 52, 234–243 (2012). https://doi.org/10.1007/s12033-012-9539-2

Download citation

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s12033-012-9539-2

Keywords

Search

Navigation