The study of chloroplast RNA splicing is usually performed by such complicated methods as Northern blot hybridization, RNAse protection or primer extension. An application of simpler RT-PCR technique may lead to underestimation of unspliced pre-mRNA. We used five protein coding genes from maize plastome to analyze factors that can reduce a pool ofintron containing transcripts as compared to a mature RNA. We revealed that a mode of DNAse inactivation, a temperature of cDNA synthesis, and a type of DNA polymerase used are important for the proper detection of unspliced RNA level. The increase in a number of PCR cycles was accompanied with a concomitant decrease in the proportion of unspliced RNA for a one gene only. We demonstrated that after PCR intron containing and intron free strands of amplicons are able to form heteroduplexes. The information obtained let us to introduce the simple and effective method for the investigation of a chloroplast spliced mRNA and unspliced pre-mRNA.