Moreover, the application of a hybrid selection step permits the isolation of rare transcripts in terms of sequence specificity. Abundant RNA molecules such as ribosomal RNAs and highly expressed mRNAs that may hinder the selective isolation of low expression transcripts are removed during the washing steps and the amplification of target molecules is facilitated. Based on the above, this protocol may prove valuable for the experimental validation of a broad spectrum of transcripts by selecting a digestion enzyme that recognizes the non-desired target. Along these lines it is conceivable that the elimination of a unique restriction site could provide the selection edge required for the isolation of a desired alternative transcript. Sequence alignment of the isolated RNase k-02 cDNA with the human RNASEK gene revealed that the RNase k-02 mRNA isoform occurs as a result of an alternative D4 donor event within the first intron, a phenomenon which is considered to be quite frequent in human gene products. It has been proposed that differential regulation of subtle alternative splicing isoforms expression levels may denote function. Particularly, the ratio between the two short-distance splice isoforms may differ in various tissues and cell types, depending on developmental stages or in response to external factors. To date, a variety of methodological implementations such as polyacrylamide and agarose gel electrophoresis, capillary electrophoresis as well as RNA sequencing analysis have been employed in order to specify the expression ratio. However, these approaches cannot provide accurate results or they are limiting due to the sophisticated equipment requirements. The Real-time PCR based methodological approach presented here may be applied for the expression analysis of variant sequences harboring distinctive restriction sites. In other words quantification, similarly to cloning is achieved in terms of the isoforms sequence differentiation. The results of our analysis demonstrated that RNase k-02 mRNA is expressed in all the examined cell lines with varying degrees of isoforms expression ratio. This observation may reflect the existence of differential regulation mechanisms that are implicated in the expression pattern of the human RNASEK gene. It is well known that apart from protein coding RNAs, an important percentage of transcription products bears no protein coding capacity. Apart from the widely studied non coding RNA populations such as rRNAs, tRNAs and microRNAs, long non coding RNAs consist a novel group of RNAs sharing a single common feature: a size of over 200 nucleotides. A large number of lncRNAs bear mRNA signatures such as 59cap and poly tail that seem to participate in their turnover. For this reason, a crucial issue in this study was to assess whether RNase k-02 mRNA isoform is protein coding or not. The isolated RNase k-02 cDNA clone contains an ORF of 405 nucleotides.