A High Resolution Short Interfering RNA (siRNA) Detection Method from Virus-infected Plants.
Citation
http://www.bio-protocol.org/e940 Vol 3, Issue 20, Oct 20, 2013
Plain language summary
In plants, RNA silencing, that is, the reduction of the levels of RNA through their destruction, acts as a natural defense mechanism against viral infection and is associated with accumulation of virus-specific small interfering RNAs (siRNAs). Novel siRNAs are continuously discovered and there is great interest in the roles these non-coding small siRNA molecules play. Therefore, there is a need for methods that can reliably detect and quantify the levels of such siRNAs in organisms. One popular and valuable analytical method to validate the presence of siRNAs is their separation on polyacrylamide gels using electrophoresis (PAGE). The so separated siRNA molecules are then transferred to and fixed to nylon membranes, followed by their detection using hybridizing radio-labeled probes. Here we describe in detail a protocol for a reliable method for the isolation and detection of small siRNAs from virus-infected plants. This method was then successfully used in our lab (Panwar et al. 2013, Panwar et al. 2013b).
Courtesy of M. Walker and H. Sanfacon.
Abstract
Plant viruses are strong inducers as well as targets of RNA silencing. In plants RNA silencing acts as a natural defense mechanism against viral infection and is associated with accumulation of virus-specific small interfering RNAs (siRNAs). The continuing discoveries, increasing awareness and interest in the regulatory roles of non-coding small RNAs have raised the need for methods that can reliably detect and quantitate the expression levels of small RNAs. Northern blot analysis of small RNAs involving the separation of RNA molecules using polyacrylamide gel electrophoresis (PAGE) has remained a popular and valuable analytical method to validate small RNAs. Northern blot analysis consist of resolving RNAs by gel electrophoresis, followed by transferring and fixing to nylon membranes as well as detecting by hybridization using radioactive probes. The following protocol provides a method for isolation and detection of small RNAs from virus-infected plants and was successfully used in Panwar et al. (2013a), Panwar et al. (2013b).