RPLP0 housekeeping gene for normalising RT-PCR assays

Updated : 2 May 2024 @ 2:10 PM

Quantitative reverse transcription PCR (RT-qPCR) is commonly used to measure gene expression because it is simple, efficient, sensitive and relatively inexpensive. However, the appropriate reference genes must be chosen carefully and measured precisely to normalise assay results. This blog post explores why the RPLP0 housekeeping gene has become a prevalent reference gene for probe-based RT-qPCR assays.    

Reference genes in RT-qPCR

To accurately verify target gene expression changes, reference genes must be chosen carefully. An ideal reference gene:

• is expressed stably in different tissues and cells,
• is expressed stably regardless of environmental, biological or abiotic stress or other factors, and
  
• has expression levels similar to the target gene.¹

Housekeeping genes regulate essential cellular functions and have relatively stable expression across all cell types regardless of differentiation type, cell cycle stage, developmental stage or tissue environment. This stability makes them reliable reference genes for RT-qPCR in many species.² Some examples include the genes for actin, tubulin, elongation factor 1-α, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and 18S ribosomal RNA.¹  

RPLP0 reference gene

One housekeeping gene has become a popular choice as a reference for RT-qPCR; the 36B4 gene encodes the acidic ribosomal phosphoprotein P0 (RPLP0), a component of the 60S subunit of the ribosome.³ RPLP0 is part of a pentameric complex that forms a stem-like structure protruding from the 60S subunit into the cytoplasm. This protrusion supports the GTPase steps in the translocation of protein synthesis, making the RPLP0 housekeeping gene a suitable target for gene expression analysis. Compared to the transcript levels of other common reference genes, such as beta-actin and cyclophilin, 36B4 (RPLP0) has proved to be a very reliable and consistent standard for gene expression analysis in multiple tissues, including brain, heart, liver, kidney, muscle and lung.⁴

The 36B4 (RPLP0) cDNA nucleotide sequence has highly conserved regions in the 5ʹ end of its open reading frame that cross tissue and species boundaries. The nucleotide sequence homology between humans and other mammals is remarkably high, with 94% homology with Canis familiaris (dog) and Bos taurus (bovine), and 99% for Pan troglodytes (chimpanzee).⁴ For the most commonly used lab animals, Mus musculus (mouse) and Rattus norvegicus (rat), the homology with the human 36B4 gene is 88% and 89%, respectively.⁵ As with other highly expressed genes, 36B4 (RPLP0) has eight related pseudo-genes in the human genome, found on chromosomes 1, 2, 3, 11, 14, 15 and 18.

Recent trends have supported using multiple reference genes as normalising factors in real-time RT-PCR.³ The choice of reference genes is tissue- and assay-specific, so no single gene is suitable for all cell and tissue types. As you design your RT-qPCR assay, the experts at LGC Biosearch Technologies™ can provide recommendations for common reference genes that may be suitable for your application.

• For more information, visit our website. 
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References 

1 Wang JJ, Han S, Yin W, et al. (2018) Comparison of reliable reference genes following different hormone treatments by various algorithms for qRT-PCR analysis of Metasequoia. Int J Mol Sci. 20(1):34. https://doi.org/10.3390%2Fijms20010034

2 Van Acker SI, Van Acker ZP, Haagdorens M, et al. (2019) Selecting appropriate reference genes for quantitative real-time polymerase chain reaction studies in isolated and cultured ocular surface epithelia. Sci Rep. 9:19631. https://doi.org/10.1038/s41598-019-56054-1.

3 Zhang WX, Fan J, Ma J, et al. (2016) Selection of Suitable Reference Genes for Quantitative Real-Time PCR Normalization in Three Types of Rat Adipose Tissue. Int J Mol Sci. 17(6): 968. doi: 10.3390/ijms17060968

4 Akamine R, Yamamoto T, Watanabe M, et al. (2007) Usefulness of the 5' region of the cDNA encoding acidic ribosomal phosphoprotein P0 conserved among rats, mice and humans as a standard probe for gene expression analysis in different tissues and animal species. J. Biochem. Biophys. Methods 70: 481-486. 10.1016/j.jbbm.2006.11.008.

5 Laborda J. (1991) 36B4 cDNA used as an estradiol-independent mRNA control is the cDNA for human acidic ribosomal phosphoprotein P0. Nucleic Acids Research. 19(14):3998. https://doi.org/10.1093/nar/19.14.3998.