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.
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
Lan
L
Sun
Joseph
J
Irudayaraj
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