Selection of housekeeping genes for use in quantitative reverse transcription PCR assays on the murine cornea

Abstract

Purpose: To evaluate the suitability of common housekeeping genes (HKGs) for use in quantitative reverse transcription PCR (qRT-PCR) assays of the cornea in various murine disease models.

Methods: CORNEAL DISEASE MODELS STUDIED WERE: 1) corneal neovascularization (CorNV) induced by suture or chemical burn, 2) corneal infection with Candida albicans or Aspergillus fumigatus by intrastromal injection of live spores, and 3) perforating corneal injury (PCI) in Balb/c mice or C57BL/6 mice. Expression of 8 HKGs (glyceraldehyde-3-phosphate dehydrogenase [GAPDH], beta-actin [ACTB], lactate dehydrogenase A [LDHA], ribosomal protein L5 [RPL5], ubiquitin C [UBC], peptidylprolyl isomerase A [PPIA], TATA-box binding protein [TBP1], and hypoxanthine guanine phosphoribosyl transferase [HPRT1]) in the cornea were measured at various time points by microarray hybridization or qRT-PCR and the data analyzed using geNorm and NormFinder.

Results: Microarray results showed that under the CorNV condition the expression stability of the 8 HKGs decreased in order of PPIA>RPL5>HPRT1>ACTB>UBC>TBP1>GAPDH>LDHA. qRT-PCR analyses demonstrated that expression of none of the 8 HKGs remained stable under all conditions, while GAPDH and ACTB were among the least stably expressed markers under most conditions. Both geNorm and NormFinder analyses proposed best HKGs or HKG combinations that differ between the various models. NormFinder proposed PPIA as best HKG for three CorNV models and PCI model, as well as UBC for two fungal keratitis models. geNorm analysis demonstrated that a similar model in different mice strains or caused by different stimuli may require different HKGs or HKG pairs for the best normalization. Namely, geNorm proposed PPIA and HRPT1 and PPIA and RPL5 pairs for chemical burn-induced CorNV in Balb/c and C57BL/6 mice, respectively, while UBC and HPRT1 and UBC and LDHA were best for Candida and Aspergillus induced keratitis in Balb/c mice, respectively.

Conclusions: When qRT-PCR is designed for studies of gene expression in murine cornea, preselection of situation-specific reference genes is recommended. In the absence of knowledge about situation-specific HKGs, PPIA and UBC, either alone or in combination with HPRT1 or RPL5, can be employed.

Figure 1

Macroscopic manifestation of various corneal disease models. Please pay close attention to the similarity or difference between related models, like corneal neovascularization induced in same animal strain by different method (S-CorNV and CB-CorNV in Balb/c mice) or induced in different strain with same method (CB-CorNV in Balb/c and C57BL/6 mice). Infection with different pathogen strains caused similar disease but with different severity (CaK and AfK in Balb/c mice).

Figure 2

Changes in expression of the 8 HKGs in murine corneas with experimental CorNV as assessed by microarray. The ratios were obtained by comparing the normalized fluorescence intensity of experimental corneas to that of the controls. In this commercial microarray, ACTB, LDHA, GAPDH, and RPL5 are used as HKGs, thus each is represented by 50 spots in the array. The average of these 50 signals was used to calculate the average and standard error for each group and this was used for comparison with the other four genes (viz. UBC, PPIA, TBP1, and HPRT1, which are represented by only one spot in the array). The data presented (mean±standard deviation) were obtained from three (for S-CorNV Balb/c D5 and CB-CorNV Balb/c D6 groups) or two (for the other three groups) arrays. The coefficient of variation (CV) was obtained by dividing the standard deviation by the mean in each model. S-CorNV Balb/c D5: suture-induced CorNV in Balb/c mice, day 5; S-CorNV Balb/c D10: suture-induced CorNV in Balb/c mice, day 10; CB-CorNV Balb/c D6: chemical burn-induced CorNV in Balb/c mice, day 6; CB-CorNV Balb/c D14: chemical burn-induced CorNV in Balb/c mice, day 14; CB-CorNV C57Bl/6 D6: chemical burn-induced CorNV in C57Bl/6 mice, day 6.

Figure 3

Raw C_t data for 8 candidate HKGs in each corneal disease model obtained using qRT–PCR. It should be noted that each point represents the mean of reactions performed in triplicate for each cDNA sample and for each gene.

Figure 4

Average expression level stability (M) and pairwise variation (V) of 8 HKGs as assessed by geNorm analysis. The genes with the lower M values are considered to have more stable expression levels. Pairwise variation was used to determine the optimal number of HKGs required for normalization. According to the algorithm and instructions provided with the software, a cutoff of 0.15 for V was used. It was apparent from analysis of all studied models that a combination of two HKGs is sufficient for normalization.

Figure 5

An example of the influence that choice of HKGs can have on the apparent changes in Tkt gene expression levels. Only data from the second time point of each treatment is shown, but is representative of that disease model, i.e., D5 for S-CorNV, D6 for CB-CorNV Balb/c and CB-CorNV C57Bl/6, D7 for CaK, AfK and PCI. The y-axis is for ratio of the Tkt expression levels in disease model corneas to that in control corneas that were obtained via direct comparison of non-normalized C_t (none) or C_t normalized using each optimal HKG or HKG pair proposed by geNorm or NormFinder, plus ACTB and GAPDH. Please note that the optimal HKGs or HKG pairs were different among models as shown in Table 4. When a HKG pair was used, the geometric means of relative expression levels against each HKG was used.