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

Table 1

Comparison of methods from current investigations regarding miRNAs in periodontal disease.

Author and year	Object of investigation	RNA extraction method	Detection method for miRNA profile	Validation of specific miRNAs	Number of study participants	Internal control	Statistical analysis	Investigated and/or deregulated miRNAs
In vitro human
Moffatt and Lamont 2011 [20]	Gingiva epithelial cells	TRIzol (Invitrogen), miRNeasy kit (Qiagen)	Microarray (LC Sciences)	qRT-PCR TaqMan (Applied Biosystems)	—	RNU-48	ANOVA, -test	miR-203
Nahid et al. 2011 [13]	Human THP-1 monocytes	mirVana miRNA isolation kit (Ambion)	—	TaqMan microRNA assay (Applied Biosystems)	—	RNU44	One-way analysis of variance followed by the two-sided, unpaired Student’s -test	miR-146a; miR-155; miR-132
Honda et al. 2012 [14]	THP-1 cells and THP-1-derived macrophages	TRIzol reagent (Invitrogen)	Agilent human miRNA microarrays (Release 12.0) (Agilent technologies)	TaqMan microRNA Assay (Applied Biosystems)	—	RNU44	Unpaired -test, ANOVA-Williams test	miR-146a; miR-155
Xie et al. 2013 [16]	Human gingival fibroblasts	TRIzol reagent (Invitrogen)	miRNA microarray analyses (Kangchen Bio-Tech)	qRT-PCR analysis (SYBR Green qPCR Master Mix PA-112, SABiosciences, Qiagen)	—	U6 small nuclear RNA	Student’s -test	miR-146a; miR-146b-5p
Sipert et al. 2014 [17]	Human fibroblasts from dental pulps, gingivae, and periodontal ligaments	TRIzol (Invitrogen)	NCode miRNA rapid labeling system (Cat. # MIRLSRPD-20) (Invitrogen) Ncode Multi-Species miRNA microarray kit V2, (Invitrogen)	Taqman miRNA assays, (Applied Biosystems)	—	U6B	Two-way ANOVA followed by Bonferroni post hoc test	miR-146a; miR-155
Naqvi et al. 2014 [18]	Human THP-1-differentiated macrophages	miRNeasy kit (Qiagen)	NanoString nCounter miRNA assay (NanoString Technologies)	Quantitative real-time PCR EvaGreen Master Mix (Biotium)	—	RNU6B	Student’s -test (two-tailed)	miR-29b; miR-32; miR-146a; miR-891
Ouhara et al. 2014 [19]	Simian virus 40 antigen immortalized gingival epithelial cell line, OBA-9	mirVana miRNA Isolation Kit (Applied Biosystem)	miRCURY LNA microRNA Array, v.16.0 (Exiqo)	TaqMan MicroRNA Assays System (Applied Biosystems)	—	RNU48	Student’s -test, Tukey honestly significant difference	miR-584
Jiang et al. 2015 [15]	Human periodontal ligament cells	TRIzol reagent (Invitrogen)	—	Quantitative RT-PCR analysis (RT SYBR® Green qPCR Master Mixes PA-112, SABiosciences, Qiagen)	—	U6 small nuclear RNA	ANOVA and Student-Newman-Keuls test	miR-146a
In vivo animal
Nahid et al. 2011 [13]	Maxillae and spleens from ApoE^−/− mice	mirVana miRNA isolation kit (Ambion)	—	TaqMan microRNA assay (Applied Biosystems)	—	snoRNU202	One-way analysis of variance followed by the two-sided, unpaired Student’s -test	miR-146a; miR-155; miR-132
In vivo human
Lee et al. 2011 [21]	Gingival tissue	mirVana™ miRNA Isolation kit (Ambion)	RT^2 miRNA PCR array system (SABiosciences)	TaqMan miRNA assays (Applied Biosystems)	n.i.	RNU44	Student’s -test	miR-181b; miR-19b; miR-23a; miR-30a; miR-let7a; miR-301a
Xie et al. 2011 [22]	Gingival tissue	TRIzol reagent (Invitrogen)	miRNA microarray (Kangchen Bio-Tech)	Quantitative RT-PCR analysis (RT SYBR Green qPCR Master Mixes PA-112, SABiosciences, Qiagen)	10 periodontitis patients; 10 healthy subjects	U6 small nuclear RNA	Unpaired Student’s -test	miR-126; miR-20a; miR-142-3p; miR-19a; let-7f; miR-203; miR-17; miR-223; miR-146b; miR-146a; miR-155; miR-205
Stoecklin-Wasmer et al. 2012 [24]	gingival tissue	TRIzol (Invitrogen); RNeasy (Qiagen,)	Microarray	qRT-PCR	198 gingival tissue samples, 158 diseased and 40 healthy samples from 86 patients with periodontitis	n.i.	R (the R Development Core Team, 2005) and bioconductor statistical frameworks	miR-451; miR-223; miR-486-5p; miR-1246; miR-1260; miR-141
Perri et al. 2012 [26]	Gingival tissue	TissueLyser LT and miRNeasy Mini Kit (Qiagen)	Quantitative microRNA PCR array; miRNA PCR Array (SABiosciences)	—	10 nonobese patients and 10 obese patients each group with 5 periodontally healthy sites and 5 chronic periodontitis sites	SNORD48 and RNU6-2	ANOVA and nonpaired Student’s -test	miR-142-3p; miR-15a; miR-30e; miR-30d; miR-22; miR-130a; miR-106b; miR-103; miR-185; miR-210; miR-18a
Ogata et al. 2014 [23]	Gingival tissue	miRNeasy Mini Kit (Qiagen)	Human miRNA microarray 8 × 15K kit (Agilent Technologies)	Real-time PCR SYBR Advantage qPCR Premix (Clontec)	3 chronic periodontitis patients and 3 edentulous residual ridges	U6	One-way ANOVA	miR-150; miR-200b; miR-223; miR-144; miR-379; miR-222
Kalea et al. 2015 [27]	Gingival tissue	mirVana miRNA Isolation Kit (Ambion)	Affymetrix GeneChip miRNA 3.0 arrays (Affymetrix)	TaqMan MicroRNA expression assays (Applied Biosystems)	36 eligible individuals	U6snoRNA and RNU6B	Partek 6.6 and 1-way analysis of variance, R-statistical environment	miR-4721; miR-557; miR-196a; miR-323a-3p; miR-200b-5p; miR-188-5p
Motedayyen et al. 2015 [25]	Gingival tissue	mirVana miRNA isolation kit (Ambion)	—	Real-time PCR (TaqMan Universal Master Mix II, no UNG, and hsa-miRNA146a kits, Applied Biosystems)	10 healthy controls, 20 chronic periodontitis patients	n.i.	Student’s -test or Mann-Whitney test; Pearson test or Spearman test	miR-146a

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 shown to draw conclusions regarding the validity of the results. n.i.: no information, only miRNAs which were validated by RT-PCR were considered, and Stoecklin-Wasmer et al. refer to an earlier report by Demmer et al. 2008 [62]. MiRNAs which were reported in more than one study are highlighted in bold type.