*5.3.5 Ribosomal intergenic spacer analysis (RISA)/automated ribosomal intergenic spacer analysis (ARISA)*

Ribosomal intergenic spacer analysis (RISA) is a simple, single-step PCR-based method for profiling microbial diversity that detects the variation in size of the intergenic transcribed spacer (ITS) region between the bacterial 16S and 23S rRNA genes [37]. RISA has been extensively used to profile microbial diversity in a range of environmental niche. Intergenic spacer (IGS) region between the 16S and 23S ribosomal subunits is amplified by PCR, denatured and separated on a polyacrylamide gel under denaturing conditions. This region encodes tRNAs and is useful for differentiating between bacterial strains and closely related species because of heterogeneity of the IGS length and sequence. In RISA, the sequence polymorphisms are detected using silver stain while in ARISA the forward primer is fluorescently labeled and is automatically detected using laser. ARISA is a rapid and effective community analysis technique which can be used in conjunction with other accurate labor-intensive methods (e.g., 16S rRNA gene cloning and sequencing) for fine-scale spatial and temporal resolution [38]. Delmont and co-workers evaluated the different DNA extraction protocols by using RISA in study of soil microbial communities and reported that the total community DNA extracted from different DNA extraction procedures generated the different RISA profiles [39].
