Sensitive detection using microfluidics technology of single cell PCR products from high and low abundance IgH VDJ templates in multiple myeloma.
Human cancer is inherently heterogeneous, so the ability to monitor individual cancer cells at every clinic visit would be a valuable tool. This work describes the first step towards developing handheld and automated devices for molecular and phenotypic analysis of cancer cells. Here, we show that use of capillary electrophoresis to detect PCR product amplified from either transcripts (high abundance template) or genomic DNA (low abundance template) encoding clonotypic immunoglobulin heavy chain VDJ of plasma cells from patients with multiple myeloma. High abundance IgH VDJ transcripts amplified in conventional systems or by capillary electrophoresis through channels on microfluidic chips or, alternatively, PCR product amplified from individual myeloma plasma cells in a single stage RT-PCR reaction was readily detectable on microfluidic chips. For low abundance templates, a nested PCR strategy was needed to detect PCR product by any method. Using microfluidic chips, PCR products amplified from genomic IgH VDJ DNA were detected in six out of eight plasma cells. Comparison of the ABI3100 and the microfluidic chip indicates that approximately 20 times more sample is injected into the ABI 3100 capillary than for the microfluidics chip. Overall, for high and low abundance template in individual cells, the microfluidic separation/detection system is at least as sensitive as the ABI 3100. In the future, integrated microfluidic platforms that incorporate both PCR cycling and product detection on the same chip are likely to exceed conventional systems in sensitivity and speed of genetic analysis by RT-PCR or PCR.
Pilarski
Linda M
LM
Department of Oncology and Cross Cancer Institute, University of Alberta, Edmonton AB, Canada. Linda.pilarski@ualberta.ca
Lauzon
Jana
J
Strachan
Erin
E
Adamia
Sophia
S
Atrazhev
Alexey
A
Belch
Andrew R
AR
Backhouse
Christopher J
CJ
D004273:DNA, Neoplasm / Q000032:analysis*; D046209:Electrophoresis, Microchip; D015894:Genome, Human; D006801:Humans; D007128:Immunoglobulin Fragments / Q000235:genetics; D007143:Immunoglobulin Heavy Chains / Q000235:genetics*; D009101:Multiple Myeloma / Q000175:diagnosis* / Q000235:genetics; D016133:Polymerase Chain Reaction; D012680:Sensitivity and Specificity; D013698:Templates, Genetic; D014158:Transcription, Genetic
D004273:DNA, Neoplasm; D007128:Immunoglobulin Fragments; D007143:Immunoglobulin Heavy Chains