title: Simultaneous quantification of alternatively spliced transcripts in a single droplet digital PCR reaction.
abstract: Human telomerase reverse transcriptase (hTERT) is an essential component required for telomerase activity and telomere maintenance. Several alternatively spliced forms of hTERT mRNA have been reported in human primary and tumor cells. Currently, however, there is no sensitive and accurate method for the simultaneous quantification of multiple alternatively spliced RNA transcripts, such as in the case of hTERT. Here we show droplet digital PCR (ddPCR) provides sensitive, simultaneous digital quantification in a single reaction of two alternatively spliced single deletion hTERT transcripts (α-/β+ and α+/β-) as well as the opportunity to manually quantify non-deletion (α+/β+) and double deletion (α-/β-) transcripts. Our ddPCR method enables direct comparison among four alternatively spliced mRNAs without the need for internal standards or multiple primer pairs specific for each variant as real-time PCR (qPCR) requires, thus eliminating potential variation due to differences in PCR amplification efficiency.
authors.affiliation: Cancer Prevention and Control Program, Lombardi Comprehensive Cancer Center, Georgetown University Medical Center, Washington, DC.
authors.forename: Bing
authors.initials: B
authors.lastname: Sun
authors.affiliation: Cancer Prevention and Control Program, Lombardi Comprehensive Cancer Center, Georgetown University Medical Center, Washington, DC.
authors.forename: Lian
authors.initials: L
authors.lastname: Tao
authors.affiliation: Cancer Prevention and Control Program, Lombardi Comprehensive Cancer Center, Georgetown University Medical Center, Washington, DC.
authors.forename: Yun-Ling
authors.initials: YL
authors.lastname: Zheng
mesh_terms: D017398:Alternative Splicing; D001483:Base Sequence; D045744:Cell Line, Tumor; D017931:DNA Primers / Q000235:genetics; D004268:DNA-Binding Proteins; D017353:Gene Deletion; D006801:Humans; D009369:Neoplasms / Q000235:genetics; D016133:Polymerase Chain Reaction / Q000379:methods*; D012333:RNA, Messenger / Q000235:genetics*; D019098:Telomerase / Q000235:genetics*
chemical_list: D017931:DNA Primers; D004268:DNA-Binding Proteins; D012333:RNA, Messenger; C509186:TERT protein, human; D019098:Telomerase
keywords: RNA alternative splicing; ddPCR; droplet digital PCR; hTERT; qPCR; real-time PCR; telomerase; telomere