Beta-2 Microglobulin And Ubiquitin C Identified as Two Robust Housekeeping Genes for RNA Expression Normalization in Real Time PCR on Human Leukocytes and Regulatory T Cells

Background : Changes in gene expression are increasingly used to evaluate the effects of exposure to environmental agents. Housekeeping genes (Hk) are essential in these analyzes as internal controls to normalize expression levels assessed in real-time PCR (RT-PCR). Ideal Hk genes (i) are constitutively expressed; (ii) do not respond to external stimuli and (iii) show small or no variation between samples or from one assay to another. Previous studies indicate that some commonly used Hk genes, including Glyceraldehyde 3-Phosphate Dehydrogenase (GAPDH) and beta-actin, have differential expression in various cell lines. The objective of this study is, first, to identify and validate the most appropriate housekeeping genes for RNA expression analysis of human primary Peripheral Blood Mononuclear Cells (PBMCs) in response to antigen like stimulation and then to verify if selected gene can be used for isolated immune population as regulatory T cells (Treg). Results : Using reverse transcriptase RT-PCR protocol, we show that following activation only B2M remain unchanged in PBMCs, which is also true regarding different donors in Tregs. UbC shows non- significant difference between donors in PBMC and isolated Treg. GAPDH and HPRT expression slightly modulate regarding PBMC donor, this small variation is also observed in Treg. In contrast, 18S and ATCB show significant variation in PBMC gene expression. Conclusion : Although our results suggest that the relevance of Hk genes should be determined for each experimental condition, B2M appear to be excellent candidate as internal controls followed by UbC.


Introduction
Housekeeping (Hk) genes are essential for quantitative gene expression techniques interpretation. By definition, a Hk gene is constitutively expressed in a stable manner in all nucleated cell types, as these genes expression are essential for cell survival and functions [1]. The Hk genes use allows to manage potential errors committed during RNA extraction or reverse transcription, as it is used as reference to normalize total RNA expression [2]. Indeed, Hk gene expression should be constant in any cell type or activation state. If not, the normalization by a variable Hk gene will lead to potential errors in gene expression quantification. Moreover, if there is a variation in Hk gene expression, small differences in genes of interest expression will not be detected [2]. However, there is no single gene known with a constant expression level in all cell type or in all activation state [3]. According to the MIQE (Minimum Information for publication of Quantitative Real-Time PCR Experiments) guidelines, the normalization should be made on more than one reference gene [4].
Many studies show that some Hk genes previously recognized as a gold standard are not as stable as thought and that Hk gene transcription can vary significantly between different donors, activation states, development stages, cell origins or experimental conditions [3]. There is no reports of stable and robust primary human PBMC Hk gene in literature . In this study, we have selected gene described in literature to be good Hk genes in different immune cell lines to test them in PBMCs. Indeed, as they are

PBMC Isolation and Culture
PBMC isolation was performed on two EDTA tubes of 8 mL from three healthy donor blood (n=3), using a density gradient with lymphocyte separation medium (Eurobio, Les Ulis, France). PBMCs

Treg Isolation
Human blood bags were collected from healthy adult donors at EFS (Etablissement Francais du sang). Treg were isolated from PBMC using Treg isolation kits (Miltenyi Biotec, Bergisch Gladbach, Germany). Obtained purity was over 70%.

Selection of Interest Housekeeping Genes
The selection of five housekeeping genes (HPRT, GAPDH, 18S, UBC, B2M) is based on the literature ( Table 2)

Real Time PCR
The RT-PCR reactions were performed, for selected Hk genes (

Statistics
The validation of expression stability was statistically tested by R and by BestKeeper methods. BestKeeper algorithm uses raw Ct values and calculates the standard variations (SD and CV). Unstable genes show SD > 1. The most stable HK gene is determined with the correlation coefficient (r) of their expression compared to the BestKeeper Index, which is the geometric mean of Ct values of the highly correlated candidate reference genes. [8,9]. R statistical analysis were done using two Way ANOVA tests.

Data Base-Analysis
The study shows data obtained in GPL570 data set from Gene Expression Omnibus (GEO). This data include 5 healthy donors, 12 patients with chronic hepatitis B and 10 hepatocellular carcinoma patients.

Results
Housekeeping gene expression levels were measured by realtime PCR on 3 healthy donors, and expressions stabilities were tested using both R and BestKeeper algorithms. The ranking of these Hk genes, from the less to the most stable is 18S, ATCB, GAPDH, HPRT, UBC and B2M. Indeed, 18S expression variation between donors is more than 3 Ct, and than 2 Ct regarding the activation state ( Figure 1). ATBC is rather stable regarding different donors as variation is less than one Ct, but there is a higher difference, more than 2 Ct, between activated and non-activated PBMCs (Figure 1). GAPDH follows the same trend with nearly no expression variation between donors but with more than 4 Ct of variation following activation ( Figure 1). In our results, the rankings of HPRT and UBC is difficult because of their very similar profiles: both are very stable between donors but shows a variation of 2 Ct between activated and non-activated conditions (Figure 1). Nevertheless, statistical analysis by 2-way ANOVA show a weakly significative difference between donors for HPRT compared to the non-significative difference for UBC. Moreover, BestKeeper analysis show power of 1.88 for UBC and of 2.26 for HPRT, as power value must be under 2 for good Hk genes, UBC seems to be better candidate (Table 1).
Finally, the most reliable Hk gene in this study is B2M. Indeed, regardless of the activation state the gene expression is very stable, without any significant differences in Ct outcome ( Figure   1). Moreover, regarding BestKeeper results, B2M is the only gene with a standard deviation under 1, as recommended (SD = 0.25 CP), and also the gene with the highest p-value [9]. Finally the power is under 2 (Power [x-fold] = 1.07) which is recommended for a good Hk gene as the efficiency of qPCR is set at 2 [9] (Table 1).  To complete these results a data base analysis has been carried out on 27 patients with different profiles regarding B2M and UBC.
Among them, 5 healthy donors, 12 patients with chronic hepatitis B and 10 hepatocellular carcinoma patients [10]. This data-based analysis confirms our results by showing a variation lower than 1Ct for B2M regardless of patients or state of health (Figure 2A).
Moreover, it also reinforces UBC results showing nearly no difference between donors but small variations between healthy donors and hepatocellular carcinoma patients (less than 2 Ct) ( Figure 2B) To go further, the 4 genes with the least significative difference between donors have been selected to be test on healthy donor primary Treg.
In this results B2M expression is stable with a difference under 1 Ct between the 9 donors ( Figure 3) with a standard deviation equal to 0.96 (Table 3). Higher variation is observed for HPRT, GAPDH and UBC with respectively standards deviation equal to 1.3; 1.61 and 1.15 (Table 3). Moreover, the power of HPRT and B2M is under 2 as it is recommended for good Hk gene (Table 3).

Discussion
Real-time PCR is a very sensitive and robust quantification method for gene expression analysis. Nonetheless, many factors can impact results, that's why robusts Hk genes are essential for reliable gene expression quantification . Besides, , as PBMCs includes different cell type, the population holds lots of various information.
It is important for us to identify strong Hk gene since PBMC are the more often used sample, easy to collect by small blood collection) for clinical trial follow-up on immune system. Large ATCB expression variation could be explained , since during activation there is a variation of the nucleo-cytoplasmic ratio [5]. The observations regarding GAPDH expression also correlate with other findings, which shows a higher variation in expression, more than 15-folds in 72 different human tissues [11]. Furthermore, the activation mechanism chooses here (anti-CD3, anti-CD28) specifically target T lymphocyte activation by mimicking immunological synapse coactivators. Because of PBMC population heterogeneity, this kind of activation could act indirectly on other cell type activation, notably by cytokine secretion.

Conclusion
Here B2M is the best candidate with stable expression level regardless of the donor and activation state. This result is reinforced by statistical data obtain with Bestkeeper and by data-