Northern Blot to Detect and Quantify Specific MicroRNA in Total RNA Extract of Plant Tissue
DEŞİFRE METNİ
MicroRNAs, miRNAs, are small, single-stranded, non-coding RNAs. miRNA incorporate into a multiprotein complex and further bind to complementary target mRNA sequences to negatively regulate gene expression.
To detect specific miRNA from total RNA from plant tissues, add a gel-loading dye containing tracking dyes and formamide — an RNA denaturing agent. Heat the sample to denature the RNA and prevent secondary structure formation prior to electrophoresis.
Load the sample and standard RNA markers into wells of a pre-assembled denaturing polyacrylamide gel. Run electrophoresis, facilitating size-based separation of RNA fragments.
Transfer the gel onto pre-wet filter papers on an electroblotting cassette. Align a buffer-soaked nylon blotting membrane over the gel. Then, stack the pre-wet filter papers. Perform electroblotting.
The electric current causes the negatively charged RNA, including the miRNA, to move out of the gel onto the positively-charged nylon membrane surface. Expose the membrane to ultraviolet light; this crosslinks the RNAs to the membrane, thereby preventing RNA loss.
Incubate the membrane in hybridization buffer containing blocking agents that occupy the non-specific binding sites to reduce the background noise.
Add a radiolabeled RNA oligonucleotide probe complementary to the target miRNA to bind specifically to it. Wash the membrane with buffer, removing unbound probes.
Use a phosphor imaging system to detect and quantify the radioactive hybridization signal intensities. The high-resolution signal indicates specific miRNA expression in the plant tissue.
