MicroRNAs as Salivary Markers for Periodontal Diseases: A New Diagnostic Approach?

Table 3

Comparison of methods for salivary miRNA diagnosis.

Author and year	Number of study participants	Form of saliva	miRNA extraction method	Detection of miRNA profile	Validation	Internal control	Statistical analysis	Potential marker for carcinoma
Park et al. 2009 [28]	50 oral squamous cell carcinoma (OSCC) patients and 50 healthy matched control subjects	Unstimulated whole saliva and saliva supernatant	mirVana™ miRNA Isolation Kit (Ambion); DNA-free™ (Ambion)	RT-preamp-qPCR (Applied Biosystems)	(i) RT-preamp-qPCR (Applied Biosystems); (ii) Four-plex RT-preamp-qPCR for miR-142-3p, miR-200a, miR-125a, and miR-93.	U6 snRNA	Mann-Whitney test	miR-125a; miR-200a
Liu et al. 2012 [31]	45 patients with OSCC and 10 patient with oral verrucous leukoplakia; 24 healthy individuals	Unstimulated saliva supernatant	mirVana PARIS Isolation kit (Ambion); DNase digestion	TaqMan miRNA assay system (Applied Biosystems)	TaqMan miRNA assay system (Applied Biosystems)	miR-16	Mann-Whitney test, Wilcoxon matched pairs test, and linear regression analysis, receiver-operating characteristics ROC analysis	miR-31
Yang et al. 2013 [33]	8 progressing LGD leukoplakias; 7 nonprogressing LGD leukoplakias; 7 healthy volunteers	Unstimulated saliva	RNeasy Micro Kit (Qiagen)	TaqMan® low density array (TLDA) qRT-PCR system (Applied Biosystems) were used for global miR expression analysis in tissue samples	TaqMan MicroRNA Assay (Applied Biosystems)	RNU6	Random variance -test, Benjamini-Hochberg false discovery rate (FDR) method, Mann-Whitney test, Student’s -tests (two-sided)	miR-10b, miR-145, miR-99b, miR-708, miR-181c, miR-30e, miR-660, miR-197
Xie et al. 2013 [36]	39 patients with esophageal cancer and 19 healthy controls	Stimulated whole saliva and supernatant	mirVana PARIS Kit (Ambion)	7 whole saliva samples for EC group and 3 for healthy group for Agilent microarray 11.0 (Agilent Technologies)	RT-qPCR SYBR Premix Ex Taq (TaKaRa)	miR-16	Mann-Whitney test or the Kruskall-Wallis test, test, receiver-operating characteristics ROC curves, Spearman’s correlation test	miR-10b, miR-144, miR-451 (in whole saliva); miR-10b, miR-144, miR-21, miR-451 (in saliva supernatant)
Matse et al. 2013 [37]	38 patients with malignant and 29 with benign parotid gland tumors	Whole saliva and supernatant	mirVana Paris kit (Ambion); DNase I (Qiagen)	TaqMan Human MicroRNA Cards (Applied Biosystems); TaqMan MicroRNA assays (Applied Biosystems)	TaqMan microRNA assays (Applied Biosystems)	U6 snRNA	Wilcoxon rank-sum test, ROC curves	hsa-miR-374, hsa-miR-222, hsa-miR-15b, hsa-let-7g, hsa-miR-132, mmu-miR-140-5p
Momen-Heravi et al. 2014 [34]	9 OSCC patients before treatment, 8 patients with OSCC in remission, 8 patients with OLP, and 9 healthy controls	Unstimulated whole saliva	RNeasy kit (Qiagen)	multiplexed NanoString nCounter; miRNA expression assay (NanoString Technologies)	TaqMan MicroRNA assay (Applied Biosystems)	miRNA-191	1-way analysis of variance, followed by a 2-tailed Mann-Whitney test or Student’s -test, ROC curves analysis	miR-27b
Salazar et al. 2014 [29]	5 HNSCC patients, 5 healthy controls for expression analysis; 56 HNSCC patients, 56 healthy controls for validation	Unstimulated whole saliva	QIAzol lysis reagent (Qiagen); NucleoSpin miRNA kit (Macherey-Nagel)	miScript™ miRNA PCR arrays (Qiagen)	RT-qPCR miScript SYBR green PCR master mix (Qiagen)	SNORD96A	Mann Withney -test, ROC curves, Wilcoxon rank sum test, Bonferroni method	miR-9, miR-134, miR-191
Zahran et al. 2015 [35]	20 healthy controls, 40 potentially malignant disorders, 20 OSCC, 20 recurrent aphthous stomatitis	Unstimulated saliva supernatant	miRNeasy serum/plasma extraction kit (Qiagen)	—	RT-qPCR SYBR green PCR kit (Qiagen)	SNORD68	One-way ANOVA, -test, Dunnett -test, Scheffe’s multiple comparison, two-tailed tests	miR-21, miR-184, miR-145
Duz et al. 2016 [32]	50 saliva samples from 25 TSCC patients (once prior to and once after surgical treatment)	Unstimulated saliva supernatant	mirVana PARIS kit (Ambion)	8 samples (4 TSCC patients 4 healthy control) using Agilent 8 × 60K human V19 miRNA microarrays	TaqMan MicroRNA assay (Applied Biosystems)	RNU6b	Two-sided Student’s -test, Receiver operating characteristic (ROC) curves, 95% confidence interval (CI)	miR-139-5p
Hung et al. 2016 [30]	20 patients with oral potentially malignant disorders, 24 healthy individuals	Unstimulated saliva supernatant	mirVana PARIS isolation kit (Ambion)	—	TaqMan microRNA assay (Applied Biosystems)	miR-16	Unpaired test, ROC curves, Kaplan-Meier method, log-rank test, Cox proportional hazard model	miR-21; miR-31

RNA extraction and isolation methods are listed. The proof of quality and quantity and quality assurance are shown as important quality criteria to draw conclusions regarding the reproducibility and standardization of the investigations. Furthermore, methods for miRNA profiling and miRNA validation are listed. In addition, the number of study participants is provided to draw conclusions regarding the validity of the results. The form of saliva is also listed.