Analysis of rare RNA transcripts using surface enhanced Raman spectroscopy

Ulhas S Kadam, Purdue University

Abstract

Gene expression (transcription) is a fundamental genetic process, in which an RNA copy is transcribed from genomic DNA in the cell nucleus. Several forms of RNA exist in eukaryotes, for example, pre-mRNA, mRNA, tRNA, rRNA, alternatively spliced mRNAs, and small RNAs. Among these RNA types alternative spliced mRNA and small RNAs are known to play significant roles in growth and development of plants. Alternative splicing is a significant process, which generates diversity of transcriptome and proteome in eukaryotic cells. It has been suggested that alternative splicing could be used as a biomarker for plant developmental processes, such as flowering, circadian rhythms, and response to pathogens. Traditionally applied techniques for analysis of gene expression suffer from lack of sensitivity and have less quantification potential. Moreover, many current approaches are not capable of multiplexing. Therefore, in this research a surface enhanced Raman spectroscopic (SERS) assay platform was designed and demonstrated to detect and quantify two alternative splice variants and two miRNA targets individually. In this research we apply a novel quantification platform based on surface enhanced Raman spectroscopy (SERS) complemented with nanotechnology to detect and quantify different RNA targets in a plant system. This also is the very first demonstration of SERS-based nanosensors in Plant Science. We will first quantify alternative splicing variants, compare the 5'- and 3'- ends transcripts numbers for TWD1 gene expression, and then demonstrate multiplex detection of miRNA and mRNAs. For analysis of alternative splicing two genes DCL2 and PTB2 were selected and these genes create several different isoforms of mRNAs. These two genes are important components of RNA biology of plants. DCL2 is involved in RNA silencing and affects the stability of plant RNAs. PTB2 is involved in trafficking on pre-mRNA from site of transcription to translational site. This study is further expanded for another question in plant transgenic mutant analysis—we present an application of SERS-based assay for detection and quantification of the TWD1 gene expression of in two T-DNA insert (twd1-2 and twd1-sup) mutant alleles and its wild type plants. In the past Raman spectroscopy was mostly been applied for qualitative studies, with selected applications biomedicine and none in the plant system. Successful attempts in developing novel controlled and reproducible SERS substrates have greatly benefited the advancement of SERS for quantitative analysis. In this research, the plasmon-phonon coupling mode was used as a self-referencing tool for the normalization of Raman spectra and quantification targets. The major advantage of the approach presented here is that, it circumvents a step on the need to reverse transcribe total RNAs and amplify them by PCR. All of the SERS experiments were validated using traditional approaches of molecular biology approaches. The SERS-based approach has huge potential to quantify genetic components (rare mRNA transcripts, DNA and RNA modifications) along with multiplexing capability at relatively low cost. This demonstrated approach can complement the molecular breeding and plant biotechnology to expedite the novel traits development in agriculture to secure food production.

Degree

Ph.D.

Advisors

Irudayaraj, Purdue University.

Subject Area

Molecular biology|Food Science|Agricultural engineering|Biophysics

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