Transcript analysis with the aid of affinity capture and gel electrophoresis on microchips

Lotta Amundsen, Nadia Del Bueno, Jari Rautio, Kristiina Iljin, Matthias Nees, Kirsi Ketola, Marko Sirkiä, Petri Saviranta, Juha Kotimaa, Mikko Pakanen, Harri Siitari, Hans Söderlund, Richard A. Mathies

Research output: Contribution to conferenceConference articleScientific

Abstract

This study focuses on quantification of selected transcription products of prostate cancer cells using a microfluidic borosilicate chip. Prostate cancer has various types that differ in the way the genes are expressed in the carcinoma cells. Some of the types are very aggressive whereas others grow slowly. Hence, a gene expression fingerprint revealing the cancer type would be very valuable for prognosis and treatment decisions. Here, the expression of selected genes in different prostate cancer cell lines was determined by TRAC1 with use of a microchip combining inline affinity capture and gel electrophoresis. TRAC (transcript analysis with the aid of affinity capture) is based on quantifying selected mRNA molecules with differently sized targetspecific oligonucleotide probes1. In conventional TRAC, the samples containing mRNAs are mixed with a hybridization solution. In this step, complementary probes, labeled with fluorescent tags, are hybridized with selected mRNAs in a stoichiometric manner. At the same time, the intrinsic polyadenylated tails of the mRNAs are hybridized with a biotinylated oligodT robe. Thereafter, the mRNAprobe hybrids are captured on streptavidincoated magnetic beads. Finally, after thorough washing, the hybridized targetspecific probes are eluted from the beads and separated by capillary gel electrophoresis. In this work, the TRAC assay was miniaturized in order to improve sensitivity and to reduce the analysis time. 5 ug aliquots of totRNA extracted from different prostate cancer cell lines were processed. After capture of mRNAprobe hybrids on streptavidincoated beads, the fluorescent probes were eluted in 10 uL and divided into 2 uL aliquots. One such aliquot (corresponding to 1 ug of totRNA) was transferred to a 4-channel microchip at a time and analyzed. First, the probes were captured in a photopolymerized affinity gel plug via complementary nucleotide sequences2. After capture, the probes were detached from the affinity plug and delivered inline to the separation channel. Finally, the probes were separated by gel electrophoresis and quantified by a laserinduced fluorescence detector. The whole procedure on chip, including capture, rinse, elution and separation, took 10 min. The capture resulted in approximately 3 to 4-fold higher signal intensities vs. injection without capture. 1 Rautio, J. J., Kataja, K., Satokari, R., Penttilä, M., Söderlund, H. and Saloheimo, M. 2006. Rapid and multiplexed transcript analysis of microbial cultures using capillary electrophoresisdetectable oligonucleotide probe pools. Journal of Microbiological Methods, 65, 404416. 2 Thaitrong, N.N., Toriello, N. M., Del Bueno, N., Mathies, R. 2009. Polymerase Chain ReactionCapillary Electrophoresis Genetic Analysis Microdevice with Inline Affinity Capture Sample Injection. Analytical Chemistry, 81, 13711377.
Original languageEnglish
Publication statusPublished - 2009
MoE publication typeNot Eligible
EventLACE2009: 15th Latin American Symposium of Biotechnology, Biomedical, Biopharmaceutical and Industrial Applications of Capillary Electrophoresis and Microchip Technology - Seville, Spain
Duration: 2 Oct 20096 Oct 2009

Conference

ConferenceLACE2009: 15th Latin American Symposium of Biotechnology, Biomedical, Biopharmaceutical and Industrial Applications of Capillary Electrophoresis and Microchip Technology
Abbreviated titleLACE2009
CountrySpain
CitySeville
Period2/10/096/10/09

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    Amundsen, L., Del Bueno, N., Rautio, J., Iljin, K., Nees, M., Ketola, K., Sirkiä, M., Saviranta, P., Kotimaa, J., Pakanen, M., Siitari, H., Söderlund, H., & Mathies, R. A. (2009). Transcript analysis with the aid of affinity capture and gel electrophoresis on microchips. Paper presented at LACE2009: 15th Latin American Symposium of Biotechnology, Biomedical, Biopharmaceutical and Industrial Applications of Capillary Electrophoresis and Microchip Technology, Seville, Spain.