MicroRNAs (miRNAs) are small RNAs (sRNAs) with approximately 21-24 nucleotides in length. They regulate the expression of target genes through the mechanism of RNA silencing. Conventional isolation and cloning of miRNAs methods are usually technical demanding and inefficient. These limitations include the requirement for high amounts of starting total RNA, inefficient ligation of linkers, high amount of PCR artifacts and bias in the formation of short miRNA-concatamers. Here we describe in detail a method that uses 80 μg of total RNA as the starting material. Enhancement of the ligation of sRNAs and linkers with the use of polyethylene glycol (PEG8000) was described. PCR artifacts from the amplification of reverse-transcribed sRNAs were greatly decreased by using lower concentrations of primers and reducing the number of amplification cycles. Large concatamers with up to 1 kb in size with around 20 sRNAs/concatamer were obtained by using an optimized reaction condition. This protocol provide researchers with a rapid, efficient and cost-effective method for the construction of miRNA profiles from plant tissues containing low amounts of total RNA, such as fruit flesh and senescent leaves.
Poor reproducibility within and across studies arising from lack of knowledge regarding the performance of extracellular RNA (exRNA) isolation methods has hindered progress in the exRNA field. A systematic comparison of 10 exRNA isolation methods across 5 biofluids revealed marked differences in the complexity and reproducibility of the resulting small RNA-seq profiles. The relative efficiency with which each method accessed different exRNA carrier subclasses was determined by estimating the proportions of extracellular vesicle (EV)-, ribonucleoprotein (RNP)-, and high-density lipoprotein (HDL)-specific miRNA signatures in each profile. An interactive web-based application (miRDaR) was developed to help investigators select the optimal exRNA isolation method for their studies. miRDar provides comparative statistics for all expressed miRNAs or a selected subset of miRNAs in the desired biofluid for each exRNA isolation method and returns a ranked list of exRNA isolation methods prioritized by complexity, expression level, and reproducibility. These results will improve reproducibility and stimulate further progress in exRNA biomarker development.
A cross between IR64 (high-yielding but drought-susceptible) and Aday Sel (drought-tolerant) rice cultivars yielded a stable line with enhanced grain yield under drought screening field trials at International Rice Research Institute. The major effect qDTY4.1 drought tolerance and yield QTL was detected in the IR77298-14-1-2-10 Backcrossed Inbred Line (BIL) and its IR87705-7-15-B Near Isogenic Line (NIL) with 93.9% genetic similarity to IR64. Although rice yield is extremely susceptible to water stress at reproductive stage, currently, there is only one report on the detection of drought-responsive microRNAs in inflorescence tissue of a Japonica rice line. In this study, more drought-responsive microRNAs were identified in the inflorescence tissues of IR64, IR77298-14-1-2-10 and IR87705-7-15-B via next-generation sequencing. Among the 32 families of inflorescence-specific non-conserved microRNAs that were identified, 22 families were up-regulated in IR87705-7-15-B. Overall 9 conserved and 34 non-conserved microRNA families were found as drought-responsive in rice inflorescence with 5 conserved and 30 non-conserved families induced in the IR87705-7-15-B. The observation of more drought-responsive non-conserved microRNAs may imply their prominence over conserved microRNAs in drought response mechanisms of rice inflorescence. Gene Ontology annotation analysis on the target genes of drought-responsive microRNAs identified in IR87705-7-15-B revealed over-representation of biological processes including development, signalling and response to stimulus. Particularly, four inflorescence-specific microRNAs viz. osa-miR5485, osa-miR5487, osa-miR5492 and osa-miR5517, and two non-inflorescence specific microRNAs viz. osa-miR169d and osa-miR169f.2 target genes that are involved in flower or embryonic development. Among them, osa-miR169d, osa-miR5492 and osa-miR5517 are related to flowering time control. It is also worth mentioning that osa-miR2118 and osa-miR2275, which are implicated in the biosynthesis of rice inflorescence-specific small interfering RNAs, were induced in IR87705-7-15-B but repressed in IR77298-14-1-2-10. Further, gene search within qDTY4.1 QTL region had identified multiple copies of NBS-LRR resistance genes (potential target of osa-miR2118), subtilisins and genes implicated in stomatal movement, ABA metabolism and cuticular wax biosynthesis.