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Simultaneous quantification of alternatively spliced transcripts in a single droplet digital PCR reaction
 
Bing Sun, Lian Tao, and Yun-Ling Zheng
Cancer Prevention and Control Program, Lombardi Comprehensive Cancer Center, Georgetown University Medical Center, Washington, DC
BioTechniques, Vol. 56, No. 6, June 2014, pp. 319–325
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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.

Humans synthesize ~150,000 different proteins from 25,000–30,000 genes by alternative splicing. It is estimated that more than 70% of human protein-coding genes produce multiple alternatively spliced mRNA transcripts (1). When these mRNAs are translated, they produce an array of proteins with diverse and even antagonistic functions. A large proportion of human genetic disorders are the result of abnormal splicing, with abnormal splicing variants thought to even contribute to the development of cancer (2, 3). Given the importance of alternative splicing in regulating cellular function, accurate quantification of multiple alternatively spliced transcripts could facilitate the discovery of new biomarkers for clinical applications and thus enhance our understanding of the role of alternative splicing in health and disease.

METHOD SUMMARY

Using a single pair of primers and two probes for the telomerase enzymatic component hTERT, four alternatively spliced mRNA transcripts (single deletions α-/β+ and α+/β-, double deletion α-/β-, and non-deletion α+/β+) are simultaneously and accurately quantified using a novel methodology based on counting data from a single droplet digital PCR (ddPCR) reaction.

Next-generation sequencing seems to be the best approach for profiling expression patterns of alternative splicing variants, but its application is limited due to its high cost and the large amount of RNA required. Specific microarrays (e.g., Affymetrix exon microarray, ExonHit) have been developed for profiling splicing variants, but these platforms cannot distinguish transcripts containing either exon A or B alone from those containing both exons.

Approximately 20 alternatively spliced variants of human telomere reverse transcriptase (hTERT) have been reported, and 4 of these transcripts (α deletion: α-/ β+; β deletion: α+/β-; α + β double deletion: α-/β-; no-deletion: α+/β+) are commonly present in most tumor tissues and may serve as specific markers for cancer diagnosis, prediction of clinical outcome, or as drug targets (4-10). The α-deletion transcript has a 36 nucleotide deletion within the conserved reverse transcriptase motif and is a dominant-negative inhibitor of telomerase activity (5). The β-deletion transcript has a 182 nucleotide deletion, leading to a truncated protein lacking the conserved reverse transcriptase motifs and resulting in a catalytically inactive telomerase (6, 7). Only the full-length hTERT transcript has been shown to be associated with telomerase activity. Telomerase activation has been shown to be associated with carcinogenesis. Over 90% of tumor tissues have increased telomerase activity comparing to adjacent normal tissues (8, 9). However, the level of telomerase activity in the tumor tissues was not correlated with hTERT transcript levels, which may be due to post-transcriptional processing resulting in the formation of alternatively spliced mRNAs. This may serve as a mechanism for regulating telomerase activity, since the relative quantities of each mRNA may determine the overall enzyme activity. Several real-time PCR (qPCR)- based assays for the enumeration of common hTERT splice variants have been developed and used to study human cancer tissues to determine splicing patterns, but these assays failed to identify a consistent correlation between the level of telomerase activity and the level of hTERT transcripts (10-14). The qPCR methods used previously have limited sensitivity of quantification for some of the alternatively spliced forms that are expressed at very low levels. In general, normal tissues were shown to express very little or no hTERT, while tumor tissues only express low levels of hTERT transcripts. Among all of the transcript variants, double deletion and α-deletion transcripts are usually the lowest, if co-expressed. Another confounding factor was the use of multiple primer pairs for each variant where multiple qPCR reactions had to be carried out together with individual internal standards. Variations in PCR amplification efficiency in this case compromise the quantification accuracy, making the comparison less reliable. Here, we demonstrated a method based on droplet digital PCR (ddPCR) for the simultaneous quantification of the four major alternatively spliced forms of hTERT using just one pair of primers in a single ddPCR reaction that results in high sensitivity without the need for internal standards.

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