Invention of polymerase chain reaction (PCR) technology by Kary Mullis in 1984 gave birth to real-time PCR. Real-time PCR - detection and expression analysis of gene(s) in real-time - has revolutionized the 21(st) century biological science due to its tremendous application in quantitative genotyping, genetic variation of inter and intra organisms, early diagnosis of disease, forensic, to name a few. We comprehensively review various aspects of real-time PCR, including technological refinement and application in all scientific fields ranging from medical to environmental issues, and to plant.

20 Although metallic elements are required for plant growth, aluminum ions (Al +3 ) can be 21 considered one of the major abiotic factors affecting productivity. In plants, the presence of 22 Al +3 can result in inhibition of root growth triggering water and nutrient deficiency. Plants 23 under stress conditions undergo gene expression changes in specific genes or post-24 transcriptional gene regulators as miRNAs that can led to resistance. In this study, we 25 investigated the miRNAs involved in the sugarcane response to aluminum stress. Four 26 miRNA libraries were generated using sugarcane roots of two contrasting (tolerant and 27 sensitive) sugarcane cultivars growing under aluminum stress to identify the miRNAs 28 involved in the sugarcane response. Here we present the first miRNAs sequencing of 29 sugarcane response under aluminum stress. The contrast of the cultivars seen in the field was 30 reflected in the micro transcriptome with opposing expression profile. We selected 394 31 differentially expressed miRNAs, in both cultivars, 22% were common between cultivars.32 Real time quantitative polymerase chain reaction was used to validate the differentially 33 expressed miRNAs through high-throughput sequencing in sugarcane roots. Target genes 34 prediction was also analyzed. Our results indicated miRNAs that modulated specific target 35 genes involved in roots development and plant aluminum stress response. Those genes can 36 be the answer to tolerance in sugarcane and used in breeding programs to develop tolerant 37 cultivars. 38 39 Introduction 40 Sugarcane (Saccharum spp.) as an important source of sugar and ethanol became the 41 third most produced commodities in the world (1.4G). In this context, Brazil figure as a 42 major sugarcane producer (500M tons) followed by India (300M), China and Thailand [1].3 43 The projections, based on the worldwide increasing demand for food and energy, are that 44 sugarcane global production will increase by 21% until 2024. Production can be increased 45 by increasing productivity and cropland expansion. The sugarcane crop expansion is evident 46 in Brazil, where nowadays more than 9.5 million ha are used to cultivate sugarcane, but the 47 demand for sugar and ethanol will increase to 10.5 million ha by the years 2023/24 [2]. 48Among the main factors that can affect agricultural productivity, soil has 49 fundamental importance since it offers not only physical support but also water and the 50 necessary nutrients for plant growth. Aluminum (Al) together with silicon and oxygen are 51 the three most abundant elements in earth crust. Although metallic elements are required for 52 plant growth, aluminum ions (Al +3 ) can be considered one of the major abiotic factors 53 affecting agriculture productivity [3]. Al is a nonessential element naturally found in the 54 soil but it is toxic and its bioavailability is highest on acidic soils (pH of 5.5 or lower), 55 resulting in inhibition of root growth, architecture alteration and elongation disruption [3].56 Around the world 50%...