Then, the SSMD for the comparison of these two groups is defined as If the two groups are independent, If the two independent groups have equal variances , In the situation where the two groups are correlated, a commonly used strategy to avoid the calculation of is first to obtain paired observations from the two groups and then to estimate SSMD based on the paired observations. Based on a paired difference with population mean and , SSMD is Statistical estimation In the situation where the two groups are independent, Zhang XHD derived the maximum-likelihood estimate (MLE) and method-of-moment (MM) estimate of SSMD. Assume that groups 1 and 2 have sample mean , and sample variances . The MM estimate of SSMD is then When the two groups have normal distributions with equal variance, the uniformly minimal variance unbiased estimate (UMVUE) of SSMD is, where are the sample sizes in the two groups and . In the situation where the two groups are correlated, based on a paired difference with a sample size , sample mean and sample variance , the MM estimate of SSMD is The UMVUE estimate of SSMD is SSMD looks similar to t-statistic and Cohen's d, but they are different with one another as illustrated in. Application in high-throughput screening assays SSMD is the ratio of mean to the standard deviation of the difference between two groups. When the data is preprocessed using log-transformation as we normally do in HTS experiments, SSMD is the mean of log fold change divided by the standard deviation of log fold change with respect to a negative reference. In other words, SSMD is the average fold change (on the log scale) penalized by the variability of fold change (on the log scale) . For quality control, one index for the quality of an HTS assay is the magnitude of difference between a positive control and a negative reference in an assay plate. For hit selection, the size of effects of a compound (i.e., a small molecule or an siRNA) is represented by the magnitude of difference between the compound and a negative reference. SSMD directly measures the magnitude of difference between two groups. Therefore, SSMD can be used for both quality control and hit selection in HTS experiments. Quality control The number of wells for the positive and negative controls in a plate in the 384-well or 1536-well platform is normally