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Small RNA discovery has been revolutionized by next-generation sequencing. Following ligation of specific linkers to small RNAs, cDNAs can be produced, which are ideally suited to sequencing using short-read platforms. Such approaches have expanded catalogue of small RNAs that are thought to act through RISC and have enabled the discovery of novel small RNA classes. Deep sequencing has also proved useful for monitoring the expression of annotated small RNAs, but alternative strategies such as microarrays and quantitative PCR (qPCR) also provide economical alternatives. Databases now offer online catalogues of known small RNAs. Northern Blot Northern blotting is one of the key techniques in molecular biology, its principle aim being the measurement of a specific messenger RNA (mRNA) [15]. The underlying principle of Northern blotting is that RNA are separated by size and detected on a membrane using hybridization probe with a base sequence complementarity, or a part, of the sequence of the target mRNA. A cDNA radioactively labelled with P-32 is the most commonly used hybridization probe. Convenient, non-radioactive detection protocols are increasingly available. A combination of anti-sense oligonucleotides as probes, together with chemiluminescence-based detection provides a rapid and simplified approach for Northern blotting [16].
Microarray Technology Although all cells of human body contain identical genetic material, the same genes are not active in every cell. Studying which genes are active and which are inactive in different cell types help scientists understand how these cells function normally and how they are affected when various genes do not perform properly. Microarray technology help researchers to learn more about many different diseases, including heart disease, mental illness and infectious diseases; to name a few. One intense area of microarray research at the National Institute of Health (NIH) is the study of cancer. With the help of microarray technology, scientists will be able to further classify the types of cancers based on the patterns of gene activity in tumor cells. Designing of treatment strategies can thus be directed to the target cell only. By examining the differences in gene activity between untreated and tumor cells, the effect of these therapy on tumours can be very well understood. Recent advances in RNAi are aiding the field of functional genomics by making loss-of-function genetic studies more tractable in numerous organisms. Although implementation of RNAi in different organisms varies, the capacity to silence genes selectively is bringing the power of loss-of-function genetics to systems was not previously amenable to such studies. There is considerable pull in the research community to generate genome-scale libraries of RNAi reagents for many organisms to allow broad screen in cultured cells. Large cell-based RNAi screens are typically done in a 96-384 well-plate format which each well contains transfection reagent, cells and an RNAi reagent for a particular gene. The recent development of cell microarrays offers the potential to accelerate high-throughout functional genetic studies. The widespread use of RNAi has prompted several groups to fabricate RNAi cell microarrays that make possible discrete, in-parallel transfection with thousands of RNAi reagents on a microarray slide. Though still a budding technolony, RNAi cell microarrays promise to increase the efficiency, economy and east of genome-wide RNAi screens in metazoan cells [41].
Quantitative PCR Because of limitations associated with microarray, researchers typically validate the results obtained by these methods using quantitative real-time polymerase chain reaction, which is considered to be the gold standard for quantifying both DNA and RNA levels. Thus, it is reasonable to expect that researchers are increasingly using qPCR as their primary assay rather than a secondary validation step, especially when ultra-high throughput is not needed. Quantitative PCR is based on quantitative relationship between the amount of starting target sample and the amount of PCR product at any given cycle number [42]. The quantitative real-time PCR measures PCR product accumulation through a dual-labeled fluorogenic probe. This method provides very accurate and reproducible quantitation of gene copies. Quantitative PCR doesn't require post-PCR sample handling, preventing potential PCR product carry-over contamination and resulting in much faster and higher throughput assays. The qPCR method has a very dynamic range of starting target molecule determination and is extremely accurate and less labour-intensive. rt-qPCR is the method of choice for accurate, sensitice and specific quantification of nucleic acid sequences. It provides a convenient and reliable tool for evaluating knockdown efficiency and functional effects of RNAi-mediated gene-silencing [43].
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