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RNA Profiling of Cell-free Saliva Using Microarray Technology

¹ School of Dentistry and Dental Research Institute, University of California-Los Angeles, 10833 Le Conte Ave., Rm. 73-017 CHS, Los Angeles, CA 90095;
² School of Medicine, UCLA;
³ Jonsson Comprehensive Cancer Center, UCLA; and
⁴ Molecular Biology Institute, UCLA;

View larger version (40K): [in a new window]   Figure 1. Detection of gene-specific RNA in cell-free saliva by RT-PCR. (A) RNA stability in saliva was tested by RT-PCR typing for ACTB after storage for 1, 3, and 6 mos (lanes 2, 3, 4, respectively). Lane 1, molecular-weight marker (100-bp ladder); Lane 5, negative control (templates omitted). (B) GAPDH (B1), RPS9 (B2), and ACTB (B3) were detected consistently in all 10 cases. Lanes 1, 2, and 3 are saliva RNA, positive control (human total RNA; BD Biosciences Clontech, Palo Alto, CA, USA), and negative controls (templates omitted), respectively.

View larger version (52K): [in a new window]   Figure 2. Amplification of RNA from cell-free saliva for microarray study. (A) Monitoring of RNA amplification by agarose gel electrophoresis. Lanes 1 to 5 are 1-kb DNA ladder, 5 µL saliva after RNA isolation (undetectable), 1 µL of 2 round amplified cRNAs (range from 200 bp to ~ 4 kb), 1 µL cRNA after fragmentation (around 100 bp), and Ambion RNA Century Marker, respectively. (B) ACTB can be detected in every main step during salivary RNA amplification. The agarose gel shows an expected single band (153 bp) of PCR product. Lanes 1 to 8 are 100-bp DNA ladder, total RNA isolated from cell-free saliva, 1st round cDNA, 1st round cRNA after RT, 2nd round cDNA, 2nd round cRNA after RT, positive control (human total RNA, BD Biosciences Clontech, Palo Alto, CA, USA), and negative control (templates omitted), respectively. (C) Target cRNA analyzed by Agilent 2100 bioanalyzer before hybridization on microarray. Only one single peak in a narrow range (50–200 bp) was detected demonstrating proper fragmentation.