The Importance Of Sample Preparation For Down Stream Applications: NGS, Sequencing, Microarray, RT-PCR, MdX, etc.

MicroRNAs are endogenous 20 to 24 nucleotide noncoding RNAs that play crucial posttranscriptional regulatory roles in plant and animals. Tremendous efforts are currently being undertaken to understand the profile of the entire miRNA population of a biological sample, which will provide useful information on miRNA activity. Many miRNA discovery tools, including micro arrays and Next-gen-based sequencing, have made it possible to comprehensively and accurately assess the entire miRNA repertoire.  This presentation deals with the importance of sample preparation on downstream applications.    A prerequisite for obtaining successful results from these approaches is an efficient method for total RNA purification without bias. The choice of the method of RNA purification is critical to the outcome of downstream analysis.  This is made more significant in variations of the plant specimens and the high phenolics, starch and other inhibitors co-isolating with the RNA.  The three most popular RNA purification methods (spin columns using Silicon Carbide, spin columns employing silica membrane and phenol/chloroform extraction) are compared in this talk in terms of quality, quantity and small RNA recovery from difficult and moderately challenging plant samples. Examples of microRNA study cases will be also discussed to highlight the importance of the RNA purification method used for different plant species.