These changes in our new method, named BFCD (Buffer Fixation and Color Development for Dot Blotting), greatly improved the performance of dot blotting. In this study, to enhance the performance of dot blotting in the detection of small RNAs, we replaced UV fixing with a fixing buffer, substituted NL membrane with NC membrane, and used color development in stead of autoradiography. For RNA detection, NL membrane is commonly used. However, it shows a very high background. NL membrane is also a commonly used supporting material with relatively high binding capacity to biological molecules. However, it has very low ability to bind with nucleic acids and proteins. NC membrane is a generally used good supporting material for many methods because of its low background. The commonly used method of fixation is ultraviolet (UV) fixing, and the commonly used membranes are nitrocellulose (NC) membrane and nylon (NL) membrane. The performance of dot blotting depends on the quality of fixing and the selection of membrane besides other factors. The traditional dot blotting procedure includes two key steps of sample fixation onto membrane and final autoradiography. However, the rapid, simple and convenient method of dot blotting for small RNA detection is relatively ignored for its low sensitivity and high background. We have also successfully developed LHCD (liquid hybridization and color development) and aLHCD (amplified LHCD) methods for small RNA detection.ĭot blotting analysis is one of the most traditional and commonly used methods to determine the presence and abundance of RNA or protein. Since the first miRNA gene was discovered in 1993, efforts by scientists have led to the improvement of several existing low-throughput technologies, such as PCR and northern blotting, to detect low levels of small RNA in a better way. However, these methods are not suitable for individual miRNA investigation. Increased interest in the roles of small RNAs has led to the development of many high-throughput methods, such as deep-sequencing and microarray, to accelerate the discovery and characterization of novel small RNAs. Compared with long RNA, small RNA has a low abundance with very small size, making it difficult to be detected using traditional methods. These tiny RNAs have revolutionized our comprehension of gene expression as key post-transcriptional regulators. Non-coding small RNAs, such as small interfering RNA (siRNA), microRNA (miRNA), and piwi-interacting RNA (piRNA), have a common feature that their sizes always fall within a narrow, defined range of between 18 and 32 nt.
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