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 × 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.

Exon-Level Expression Profiling: A Comprehensive Transcriptome Analysis of Oral Fluids

“…These steps were followed with a final extension of 5 min at 72°C and cooling to 4°C. 9 All microarray raw data have been deposited in the Gene Expression Omnibus (GEO) database under series accession no. GSE7760 (http://www.ncbi.nlm.nih.…”

“…Saliva contains clinically discriminatory RNA and protein biomarkers of oral cancer (6 ), Sjögren syndrome (7 ), and sleep deprivation (8 ). As with archived and postmortem clinical samples, however, the low concentrations and partially fragmented and degraded nature of RNA in saliva pose challenges to microarray-based transcriptome profiling and validation via quantitative PCR (qPCR) 8 (9,10 ). The prevailing T7 in vitro transcription (IVT) amplification method (11 ) either depends solely on the existence of a poly(A) tail or uses random priming, which severely shortens transcripts and may lead to the overrepresentation of long transcripts.…”

Salivary mRNA targets for cancer diagnostics

“…This work also demonstrated the unequivocal conclusion that the salivary RNA signals detected can neither originate from contaminating genomic DNA nor from pseudogenes, as none of the 117 sequences obtained matched the sequences of known pseudogenes or could originated from the DNA sequence of the gene [21]. Also importantly all transcripts identified by sequencing are fromhuman origin.…”

“…Since our initial reports we have published and accumulated an abundance of additional supporting evidence, ranging from the characterization of salivary RNA [13], over assessing salivary RNA integrity by cDNA library analysis [21], to identifying and supporting the commercialization of an effective stabilizer of RNA in whole saliva, RNAprotect® Saliva (QIAGEN) [22]. Salivary cDNA library analysis showed that none of the 117 cloned mRNAs from saliva supernatant are from a pseudogene of contaminating genomic origin.…”

Current potential and limitations of molecular diagnostic methods in head and neck cancer

“…Park et al [70] have previously shown that a transcriptome is found in saliva, and subpanels of these mRNAs can be used as oral cancer biomarkers. In 2009, Park et al [71] measured the presence of microRNAs (miRNA) in saliva and determined their potential as an additional set of oral cancer biomarkers.…”