Estimation of the Variance Components in TP53 mRNA Expression in the Rat Lens after in vivo Exposure to Ultraviolet Radiation B

The aim of this study was to investigate the variance components in TP53 mRNA expression after in vivo exposure to double threshold dose of ultraviolet radiation B (UVR-B). Polymerase chain reaction (PCR) is a modern method of estimation of the gene expression in the tissue [1]. There are several types of PCR. Quantitative PCR is the type of PCR that measures quantity of the target DNA or RNA. Reverse transcription PCR is the type of PCR that detects RNA expression. The method of PCR was invented by a Nobel Prize-winning American scientist, Kary Mullis, in 1983 [2]. It is a universal tool for both diagnostic and scientific purposes [3, 4].

The quantitative RT-PCR represents several steps in its process. First, RNA is extracted from the tissue. Second, cDNA is synthetized from the RNA. Third, a quantitative RT-PCR is run using the cDNA and primers with fluorescing probe of choice.

TP53 gene is the gene that encodes transcription factor protein p53. The p53 involves in several cell processes such as repair of genotoxic damage, senescence, cell survival, and apoptosis [5]. Thus, p53 is an important protein in cell life. The p53 is expressed in the lens cells after in vivo exposure to UVR-B and plays a crucial role in apoptosis of the lens cells [6, 7]. The p53 is expressed differently in different regions of the lens after in vivo exposure to UVR-B; in the lens cortical fiber cells, it is not expressed, while in the lens epithelial cells, there is an increased expression of the p53 [8].

It is of need of biomedical researchers to be able to detect maximum signal of choice with minimum variation in the biological sample. There are different detection methods in biomedical research; one of them is a quantitative RT-PCR. In this research article, author investigates the variation of parameters in the quantitative RT-PCR of TP53 mRNA expression in the lens cells after in vivo exposure to UVR-B. So, the purpose of the study was to investigate the variance components in TP53 mRNA expression after in vivo exposure to double threshold dose of UVR-B.

The experimental animal was the six-week-old albino Sprague-Dawley female rat (Taconic, Denmark). All animals were treated in accordance with the ARVO Statement for the Use of Animals in Ophthalmic and Visual Research and ARRIVE guidelines. The Uppsala Ethics Committee on Animal Experiments, protocol number C29/10, gave the ethical approval.

An UVR-B at 300 nm was generated by the high-pressure mercury arc lamp (model 6828; Oriel, Stratford, CT, USA). A double monochromator has given the radiation centered at 300 nm. The emerging radiation had a spectral distribution centered at 300 nm with dual peaks at 297.5 nm and 302.6 nm with 10.2 nm full width at half maximum [9]. The thermopile (model 7101; Oriel, Stratford, CT, USA) calibrated to a US National Institute of Standard traceable source measured the intensity of UVR.

The animal was anesthetized intraperitoneally by a mixture of 90 mg/kg ketamine and 10 mg/kg xylazine 15 min before the exposure. Thereafter, a rat restrainer [10] held the animal, and tropicamide 10 mg/mL was instilled in both eyes to induce mydriasis. One eye of each animal was exposed to a double threshold dose of 8 kJ/m² of UVR at 300 nm for 15 min [11], while the contralateral eye was shielded during the exposure [12].

The carbon dioxide asphyxiation, followed by cervical dislocation, was the sacrifice method. The eyes were enucleated, and the lenses were extracted. The remnants of the ciliary body were removed from the lens equator under a microscope, keeping the lens in balanced salt solution (BSS; Alcon, Fort Worth, TX, USA).

NucleoSpin RNA II (Macherey-Nagel GmbH & Co., Duren, Germany) was the RNA isolation kit. The PCR using p53 DNA-specific primers, forward, 5′-ACC​CTC​TGA​CCT​TTT​TCC​CA-3′ and reverse, 5′-TGC​TGG​GAT​CTT​AGG​CAC​TC-3′ (biomers.net GmbH, Ulm, Germany), and Taq DNA polymerase (dNTPack) checked the sufficient removal of DNA from the analyte. The expected PCR product was 243 base pairs. None of the analytes revealed any DNA specific PCR products on 1.5% agarose gel electrophoresis. The RNA concentration in the analyte was measured as absorbance in a NanoDrop ND-1000 spectrophotometer (NanoDrop Products, Wilmington, DE, USA), and the cDNA was synthetized using 1 μg of total RNA by 1st strand cDNA synthesis kit, Roche Diagnostics GmbH, Mannheim, Germany.

The cDNA made from lens RNA was analyzed in triplicates by quantitative real-time PCR on an iCycler MyiQ Single-Color Real-Time PCR detection system (Bio-Rad Laboratories, Hercules, CA, USA). The TaqMan Gene Expression Master Mix (Applied Biosystems, Foster City, CA, USA) was used with the TaqMan assays for p53 (Rn00755717_m1) and 18s (Hs99999901_s1), according to the manufacturer’s instructions. Primary fluorescence measurements were fitted with the MyiQ (Bio-Rad Laboratories, Hercules, CA, USA) algorithm, and threshold fluorescence was selected and standardized for each PCR plate by the algorithm. The number of cycles at threshold fluorescence was used as the measurement in MyiQ software. The PCR efficiency was estimated to 90%.

First, a fraction of cDNA from three randomly chosen lenses was serially diluted. The serial dilutions were run together with the cDNA from samples. Second, a standard curve, expressing the number of cycles at threshold as function of relative concentration, was established for the serial dilutions in each plate. Third, the threshold number of cycles for each sample was compared to the standard curve to obtain the relative concentration of the sample cDNA measured.

Altogether, 12 rats were used in the experiment. One eye in each animal was exposed in vivo to UVR-300 nm. Animals were sacrificed at 1, 3, 8, and 24 h after exposure to UVR-B, 3 animals in each group distributed in 1 h group, 3 h group, 8 h group, and 24 h group. Samples from all lenses were processed for quantitative RT-PCR of TP53 mRNA. Additionally, the 18s rRNA was used as a reference for each lens sample. Finally, the fold change in mRNA expression of the TP53 between exposed and nonexposed lenses was calculated. Analysis of variance was performed where groups, animals, and measurements were parameters.

The significance level and the confidence coefficient were set to 0.05 and 0.95, respectively, considering the sample size.

The variance for groups is estimated to be 0.15 rel.². There is no difference among the groups (test statistic = 2.1, F = 4.06). The variance for animals is estimated to be 0.29 rel.². The variance for measurements is estimated to be 0.32 rel.².

The estimated sample size is 33 rats when the number of measurements is 6, a significance level is 0.05, a power is 0.8, a minimum detectable difference in TP53 mRNA expression is 0.3, and variances for animals and measurements are above calculated. The estimated sample size is 71 rats when the number of measurements is 6, a significance level is 0.05, a power is 0.8, a minimum detectable difference in TP53 mRNA expression is 0.2, and variances for animals and measurements are above calculated.

The study was designed to investigate the variance components in TP53 mRNA expression after in vivo exposure to double threshold dose of UVR-B. The dose 8 kJ/m² of UVR at 300 nm was selected to be a double threshold dose [13] that induces significant forward lens light scattering [14] and causes expression of apoptotic markers p53 and caspase 3 on mRNA [7] and protein [6] levels.

The time intervals of 1, 3, 8, and 24 h after the exposure were chosen based on previous investigation [15] that finds a peak of TUNEL-positive staining of epithelial cell nuclei in the rat lens at 24 h after in vivo exposure to UVR-B at 300 nm. So, with this study, the author aimed to study the time interval just before the 24 h postexposure. In time evolution, TP53 is expressed before the TUNEL-positive staining of epithelial cell nuclei.

The finding that there was no difference among the groups could be explained by the close standing time intervals to each other. Both the variance for animals of 0.29 rel.² and the variance for measurements of 0.32 rel.² show that the variance for animals is in the same order as the variance for measurements. It means that the variation level given by animals is principally the same as the variation level given by measurements, which results in implication that one can change both variances in order to minimize them for better precision of the method, in this case qRT-PCR. It is of interest that variance is the square of the standard deviation; thus, variance is defined in terms of the deviations of the observations from the mean that are squared, which gives the variance of a nonnegative number [16]. In order to minimize variance, it is necessary to understand the composition of the variance. For example, variations given by animals lie mostly in a biological difference between animals, whereas variations given by measurements lie in pipetting skills of the person and a technological (measuring and processing machines and equipment, batch belonging to the molecular products) variability. Improving the pipetting skills and strictly controlling the technological process will minimize the variance for measurements, whereas a biological difference between animals will not be possible to improve in order to decrease the variance for animals except for the same age, gender, race, and housing conditions which were controlled during this experiment as described in the article by Galichanin et al. [12].

In order to detect minimum difference in TP53 mRNA expression of 0.2, 71 rats are required in sample size. It is a large sample size. Reduction is one of three principles of humane use of animals in scientific research. It means that scientists need to reduce the number of animals in order to obtain information from fewer animals in scientific research [17]. In order to achieve this, one needs to minimize the variances. It is consequently to use fewer animals in a sample size.

Thirty-three rats in sample size are required to detect minimum difference in TP53 mRNA expression of 0.3. The sample size of 33 rats is a sample size that ranges between small and large sample sizes. The sample size of 30 is a boundary between small and large sample sizes. Although sample size of 33 rats is double less than sample size of 71 rats, the detection of minimum difference in TP53 mRNA expression is neglected. The minimization of the variances is necessary in order to achieve the acceptable level of detection of the difference in TP53 mRNA expression.

Variances are an important statistical component in scientific research that originates from different sources during the experiment. In this experiment, the sources of variation in the experiment are shown, and the variances are calculated. It was done in order to obtain the necessary sample size and to detect the minimum difference in TP53 mRNA expression. Good knowledge of statistics is necessary to have in order to design the experiment carefully. This experiment can serve as a basis in designing future scientific studies on mRNA expression.

The variance for measurements is on the same order as the variance for animals in TP53 mRNA expression in the rat lens after in vivo exposure to UVR-B, and the minimization of the variance for measurements is needed in order to obtain the acceptable level of detection of the difference in TP53 mRNA expression and the reduction in the sample size.

Ethical approval was obtained from the Uppsala Ethics Committee on Animal Experiments, protocol number C29/10.

The author declares that he has no competing interests.

This work was funded by the Karolinska Institutet Eye Research Foundation (providing the materials, collection of the data), Gun och Bertil Stohnes Stiftelse (providing the materials, collection of the data), Swedish Society of Medicine (providing the materials, collection of the data), and Karin Sandqvists Stiftelse (presentation of the data at the congress).

The author Galichanin Konstantin has designed the study, performed the study, analyzed the results of the study, and drafted and reviewed the manuscript. The paper was presented as an abstract at the European Association for Vision and Eye Research Congress 2019.

All data generated or analyzed during the study are included in this published article. Further inquiries can be directed to the corresponding author.