The high throughput RNA sequencing (RNA-seq) technology has become the popular method of choice for transcriptomics and the detection of differentially expressed genes. Sample size calculations for RNA-seq experimental design are an important consideration in biological research and clinical trials. Currently, the sample size formulas derived from the Wald and the likelihood ratio statistical tests with a Poisson distribution to model RNA-seq data have been developed. However, since the mean read counts in the real RNA-seq data are not equal to the variance, an extended method to calculate sample sizes based on a negative binomial distribution using an exact test statistic was proposed by . In this study, we alternatively derive five sample size calculation methods based on the negative binomial distribution using the Wald test, the log-transformed Wald test and the log-likelihood ratio test statistics. A comparison of our five methods and an existing method was performed by calculating the sample sizes and the simulated power in different scenarios. We first calculated the sample sizes for testing a single gene using the six methods given a nominal significance level α at 0.05 and 80% power. Then, we calculated the sample sizes for testing multiple genes given a false discovery rate (FDR) at 0.05 and 0.10. The empirical power and true prognostic genes for differential gene expression analysis corresponding to the estimated sample sizes from the six methods are also estimated via the simulation studies. Using the sample size formulas derived from log-transformed and Wald-based tests, we observed smaller sample properties while maintaining the nominal power close to or higher than 80% in all the settings compared to other methods. Moreover, the Wald test based sample size calculation method is easier to compute and faster in an RNA-seq experimental design. Later, several sample size calculation methods that were derived from the score statistic and the log-likelihood ratio test (LRT) statistic using the Poisson distribution were proposed [16]. However, the assumption of a Poisson distribution that the expected mean and variance are equal usually does not hold for RNA-seq studies, where the variance is typically greater than the mean of the read counts [17]. Therefore, a negative binomial distribution with a dispersion parameter is used to model RNA-seq data by the existing software packages such as DESeq [17] and edgeR [18], in which an exact test is used to test DEGs between conditions. Subsequently, a sample size calculation method based on an exact test statistic with the aid of the edgeR package [18] was proposed [19]. However, sample size methods derived from other test statistics such as the Wald test, the LRT and an extension of Wald test via log-transformation using negative binomial distribution to model the RNA-seq data have not yet been explored.