PCR-free quantification of multiple splice variants in a cancer gene by surface-enhanced Raman spectroscopy.
We demonstrate a surface-enhanced Raman spectroscopy (SERS) based array platform to monitor gene expression in cancer cells in a multiplex and quantitative format without amplification steps. A strategy comprising DNA/RNA hybridization, S1 nuclease digestion, and alkaline hydrolysis was adopted to obtain DNA targets specific to two splice junction variants, Delta(9,10) and Delta(5), of the breast cancer susceptibility gene 1 from MCF-7 and MDA-MB-231 breast cancer cell lines. These two targets were identified simultaneously, and their absolute quantities were estimated by a SERS strategy utilizing the inherent plasmon-phonon Raman mode of gold nanoparticle probes as a self-referencing standard to correct for the variability in surface enhancement. The results were then validated by reverse-transcription polymerase chain reaction. Our proposed methodology could be expanded to a higher level of multiplexing for quantitative gene expression analysis of any gene without any amplification steps.
Sun
Lan
L
Department of Agricultural and Biological Engineering, Bindley Bioscience Center and Birck Nanotechnology Center, Purdue University, 225 S. University Street, West Lafayette, Indiana 47907, USA. sun22@purdue.edu
Irudayaraj
Joseph
J
D017398:Alternative Splicing / Q000235:genetics*; D001483:Base Sequence; D001943:Breast Neoplasms / Q000235:genetics*; D045744:Cell Line, Tumor; D005260:Female; D015972:Gene Expression Regulation, Neoplastic; D006046:Gold / Q000737:chemistry; D006801:Humans; D053768:Metal Nanoparticles / Q000737:chemistry; D009693:Nucleic Acid Hybridization; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015719:Single-Strand Specific DNA and RNA Endonucleases / Q000378:metabolism; D013059:Spectrum Analysis, Raman / Q000379:methods*
D006046:Gold; D015719:Single-Strand Specific DNA and RNA Endonucleases