Small RNA extraction and expression analysis by Northern blot.

Small non-coding RNAs are important regulatory elements able to cause endogenous gene silencing at both the transcriptional and post-transcriptional levels. Here we describe in detail the procedure of small RNAs extraction from grape tissues, which was developed as a modification of an RNA extraction method initially used for pine tree tissues. Polyacrylamide gel electrophoresis in denaturing conditions (obtained with high concentrations of urea) is used to efficiently separate small RNAs. In hybridization of small RNA northern blot, a single stranded DNA or LNA oligonucleotide in antisense orientation to the target small RNA is used as probe. The technique reported here was used for the isolation and expression analysis of small RNAs from grape berries at different stages of maturation and allowed the identification of several non-conserved miRNAs and siRNAs. Some major constraints of the procedure are discussed, including the comparably high amounts of tissues needed for the isolation of small RNAs, especially when berries are used as samples. Some recently developed methods to address these problems are briefly discussed, which are based on Real-Time (or quantitative) PCR.