Characterization of salivary RNA by cDNA library analysis

Abstract

Oral fluid (saliva) meets the demands for a noninvasive and accessible diagnostic medium. Recent reports by our group and others described the presence and use of human RNA in saliva as a diagnostic or forensic tool, including the use for oral cancer detection. To gain insights into the integrity of salivary RNA, we examined in detail the integrity of salivary RNA by generating a cDNA library from pooled supernatant saliva of 10 healthy donors. From a library with a primary library titer of 1.3 x 10(6) cfu/mL of which 95% of the clones had inserts, we successfully sequenced 117 random colonies containing recombinant clones. BLAST search results indicated that all of these clones contained sequences of human origin. Most of the salivary RNAs appeared to be endonucleolytically cleaved at random positions as indicated by comparisons to respective full length parental RNAs from the Genbank. Twelve of the insert sequences matched to the normal salivary core transcriptome sequences, which are highly abundant mRNAs present in healthy individuals. This study provides an in-depth molecular analysis of the saliva transcriptome and should be a useful resource for future basic and translational studies of RNA in human saliva. In addition, this paper presents unequivocal evidence for the presence of RNA in saliva as determined by the use of diverse techniques such as reverse transcriptase quantitative polymerase chain reaction (RT-qPCR), in vitro translation, and the construction of a salivary cDNA library.

Figure 1

Virtual gel derived from Agilent Bioanalyzer Analysis. Shown are electropherograms of RNA extracted from saliva (A) and MCF-7 breast cancer cells (B) before and after treatment with DNase or RNase as described in the materials and methods section. The salivary RNA was concentrated 10x before it was run on a Picochip. The two peaks that were observed in the Agilent profile of saliva are indicated with a * and ** on the right side of the saliva electropherogram. The cell line RNA was run on a Nanochip.

Figure 2

A. RT-qPCR amplification plots for RT+ and RT- reactions of IL-1β, IL-8 and β-actin mRNA. B. In vitro translation of saliva RNA. Lane 1 – untreated salivary RNA. Lane 2- salivary RNA treated with DNase I. Lane 3- salivary RNA treated with RNase cocktail. The product of each in vitro translation reaction was run on an 8-16% SDS-PAGE protein gel.

Figure 3

Degradation status of human mRNA in saliva. Results of bidirectional sequencing of colonies containing nucleus-encoded human mRNA sequences (total of 62 clones). Length of each cDNA insert is expressed to its respective full length parental mRNA. The positions of each bar represents the location of cDNA inserts on their respective full length parental mRNA. Arrows (left) indicate 3 cDNA clones that are >90% full length.

Figure 4

Correlation between the length of salivary mRNA to the respective full length parental mRNA. The relative length of salivary mRNAs (cDNA inserts) are plotted against its respective full length parental mRNA.