Targeted transcript quantification in single disseminated cancer cells after whole transcriptome amplification

Abstract

Gene expression analysis of rare or heterogeneous cell populations such as disseminated cancer cells (DCCs) requires a sensitive method allowing reliable analysis of single cells. Therefore, we developed and explored the feasibility of a quantitative PCR (qPCR) assay to analyze single-cell cDNA pre-amplified using a previously established whole transcriptome amplification (WTA) protocol. We carefully selected and optimized multiple steps of the protocol, e.g. re-amplification of WTA products, quantification of amplified cDNA yields and final qPCR quantification, to identify the most reliable and accurate workflow for quantitation of gene expression of the ERBB2 gene in DCCs. We found that absolute quantification outperforms relative quantification. We then validated the performance of our method on single cells of established breast cancer cell lines displaying distinct levels of HER2 protein. The different protein levels were faithfully reflected by transcript expression across the tested cell lines thereby proving the accuracy of our approach. Finally, we applied our method to breast cancer DCCs of a patient undergoing anti-HER2-directed therapy. Here, we were able to measure ERBB2 expression levels in all HER2-protein-positive DCCs. In summary, we developed a reliable single-cell qPCR assay applicable to measure distinct levels of ERBB2 in DCCs.

Interleukin-6 trans-signaling is a candidate mechanism to drive progression of human DCCs during clinical latency

“…Gp130 forward primer was 5′-GGA CCA AAG ATG CCT CAA CT-3′ and reverse primer was 5′-GGC AAT GTC TTC CAC ACG A-3′, with an annealing temperature of 58°C and an amplicon size of 280 bp. gp130 expression was assessed by quantitative PCR (qPCR) on reamplified and purified (Qiagen PCR Purification Kit, Qiagen, Germany) WTA products of single cells 74 . To normalize for the template input, quantification of yields in the individual samples was spectrophotometrically conducted using the NanoDrop 2000 instrument.…”

“…To normalize for the template input, quantification of yields in the individual samples was spectrophotometrically conducted using the NanoDrop 2000 instrument. The DNA input for each qPCR of a single cell was normalized to 2.5 ng and the qPCR run 74 with the following primers: gp130 forward primer, 5′-ATA TTG CCC AGT GGT CAC CT-3′ and reverse primer, 5′-AGG CTT TTT GTC ATT TGC TTC T-3′, with an annealing temperature of 58°C and an amplicon size of 125 bp. Fold changes in gp130 expression were calculated from the delta Cp values between MCF-10A or MCF-7 cultured with and without MSCs.…”

“…gp130 expression was assessed by quantitative PCR (qPCR) on reamplified and purified (Qiagen PCR Purification Kit, Qiagen, Germany) WTA products of single cells 74 . To normalize for the template input, quantification of yields in the individual samples was spectrophotometrically conducted using the NanoDrop 2000 instrument.…”

Interleukin-6 trans-signaling is a candidate mechanism to drive progression of human DCCs during periods of clinical latency

“…Whole transcriptome amplification of single cells or undissociated spheres was performed as previously described 19,74 . The quality of WTA products was assessed by expression analysis of three housekeeping genes: EEF1A1, ACTB and GAPDH.…”

“…Gp130 forward primer was 5'-GGA CCA AAG ATG CCT CAA CT -3', reverse primer 5'-GGC AAT GTC TTC CAC ACG A -3', annealing 58°C and amplicon size 280 bp. gp130 expression was assessed by quantitative PCR on re-amplified and purified (Qiagen PCR Purification Kit, Qiagen, Germany) WTA products of single cells as previously described74 . To normalize for the template input quantification of yields in the individual samples was spectrophotometrically conducted using the NanoDrop 2000 instrument.…”