Quantification of alternatively spliced transcripts by Real-time PCR using intercalating dyes

Abstract

Alternative splicing of primary RNA transcripts provides the basis for a tremendous variety of the transcriptome and represents an essential instrument to generate proteins with modified properties from a single gene. In single cells, the number of splice variants is tightly regulated and needs to be determined since their ratio controls their cellular functions. Respecting the MIQE guideline [1], we have developed strategies to quantify alternatively spliced mRNA species using a combination of one-step quantitative Real-time PCR (one-step qRT-PCR) and high resolution melt (HRM) analysis of the PCR-products. To determine alternatively spliced transcripts we designed PCR-primers i. overlapping the splice site, ii. flanking the spliced region and iii. located within the excised exon and we compared their amplification products with iv. amplicons obtained with primer pairs located outside of the spliced region. In a straightforward approach, we used the common intercalating dyes SYBRGreen and SYBRGreener for detection. The specificity of the primers for a given variant was verified by Melting curve- and HRM- analysis of the PCR products as well as by gel electrophoresis. The reliability of the data was tested using mixtures of cDNA templates in different molar ratios. We found that the different strategies showed uniform results and provide powerful alternatives to quantify spliced transcripts. We used our method to measure the abundance of cation channel variants in total RNA from brain as well as from pancreatic ß- and pituitary gland cells. Variants lacking 18 amino acid residues, that do not show any channel activity after stimulation and which build channel complexes with functional variants, were found to be expressed in significant amounts of 12-24 % within these tissues and cells. We conclude that their expression might serve as a mechanism to modulate cation entry into the cell.