RNA interference (RNAi) is a gene silencing technology that involves sequence-specific silencing of target genes driven by non-coding RNAs (ncRNAs). The roles of RNAi in crop improvement have been illustrated in plant biomass regulation, enhancement of crop yield and productivity, development of seedless fruits, shelf-life enhancement, secondary metabolite regulation, nutritional improvements, enhanced biotic (bacteria, fungi, viruses, nematodes, insects) and abiotic stress tolerance (drought, salinity, cold, etc.). Advances in high-throughput techniques have paved the way for a new generation of various omics approaches such as genomics, transcriptomics, proteomics and metabolomics, which played an important role in crop science. The increasing use of NGS and high-throughput gene expression and quantification technologies over the last two decades has increased the published data stored in public repositories which triggered an explosion of research studies available through public databases. This stored information in public databases offers an invaluable resource for meta-analysis to reuse these data and generate new scientific findings. Consequently, there has been an enormous advancement in the number of systematic reviews and meta-analyses over the past decade. This chapter highlights the importance of next-generation sequencing technology, various bioinformatics methods and meta-analysis in understanding RNAi phenomena and their role in crop improvement. Additionally, this chapter also elaborates on the application of gene expression/meta-analysis in animals and plants and the workflow used in a common meta-analysis experiment, highlighting the key points that the researcher has to consider while conducting the analysis.