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High-yield RNA-extraction method for saliva.

Human saliva has been used as an auxiliary biological fluid for disease diagnosis (1-3), because the biomolecular composition of saliva changes in a disease state. Total RNA extracted from saliva is useful in determining gene expression profiles in cancer, as well as in genetic studies (4, 5). Salivary RNA is prone to degradation by RNases and other nucleases present in saliva, and therefore care should be taken to alleviate or minimize sample degradation before downstream transcriptomic analysis. Although the number of methods for isolating RNA from saliva has markedly increased over the years, most of the protocols remain relatively costly, owing to the predominant use of commercial kits (6) and, more importantly, the relatively low RNA yields. Furthermore, a large body of literature on transcriptomic studies of saliva has considered only the cell-free salivary supernatant as a biological source for isolating RNA, as opposed to the salivary cellular pellet (7-10). New high-throughput RNA-isolation methods have emerged, but these methods were developed to isolate RNA from the cell-free salivary supernatant; they also require relatively large volumes of saliva. Therefore, there is a need for a high-throughput and cost-effective method for isolating high yields of RNA (from both the cell-free salivary supernatant and the cell pellet) from small saliva volumes for identifying genes that orchestrate tumor initiation and progression. For example, epithelial cells lining the oral cavity of patients with oral squamous cell cancer undergo malignant cellular transformations within the oral cavity that are reflected at the gene level. Interrogating gene expression in only the cell-free salivary supernatant might miss these molecular events. The goals of our study were 3-fold: first, to develop an in-house highRNA yield method that uses the QIAzol lysis reagent (Qiagen) to isolate RNA from both the cellular pellet and the cell-free salivary supernatant; second, to compare results obtained with the QIAzol lysis reagent method (hereafter referred to as the QIAzol method) with results obtained with a commercial kit (NucleoSpin[R] RNA II kit (Macherey-Nagel); and third, to validate the QIAzol method in saliva samples collected within a clinical setting (i.e., in a cohort of cancer patents for whom saliva production and secretion are significantly reduced owing to chemotherapy and/or radiation treatments).

The study was approved by the University of Queensland Medical Ethical Institutional Board and the Princess Alexandra Hospital, Metro South Health Service District Human Ethics Committee. All participants gave informed consent before sample collection. Whole-mouth saliva was collected from healthy, resting control individuals (n = 9) and patients (n = 8), as previously described (11-14). Saliva was collected in DNase- and RNase-free Falcon tubes (50 mL), frozen immediately on dry ice, and stored at -80 [degrees]C until further analysis.

For the QIAzol method, saliva samples were centrifuged at 11 000g for 20 min at 4 [degrees]C to separate supernatant from the cellular fraction. RNA was extracted from both the cell pellet and the cell-free supernatant (200 [micro]L) by adding 800 [micro]L QIAzol. The samples were briefly vortex-mixed and incubated for 5 min at room temperature. Then, 200 [micro]L chloroform was added to each 2-mL Eppendorf tube, and the tube was vortex-mixed vigorously and incubated a second time for 5 min at room temperature. The samples were then centrifuged at 10 000g for 10 min at 4 [degrees]C. The upper aqueous layer (800 [micro]L) was transferred to a new Eppendorf microcentrifuge tube, 200 [micro]L chloroform was added to the Eppendorf tube, and the tube was vortex-mixed. The samples were then centrifuged at 10 000g at 4 [degrees]C. The upper aqueous layer (800 [micro]L) was transferred to a new Eppendorfmicrocentrifuge tube, an equal volume of isopropyl alcohol was added, and the samples were incubated on ice for 10 min. The samples were then centrifuged at 10 000gfor 20 min, and the supernatant was removed and discarded. The pellet was washed with 1 mL 70% molecular-grade ethanol and centrifuged at 10 000g for 5 min at 4 [degrees]C. The pellet was air dried, resuspended in 20 [micro]L RNase-free water, and stored at -80 [degrees]C. RNA aliquots were treated with RNase-Free DNase (Qiagen) in accordance with the manufacturer's protocol.

We compared our in-house QIAzol method to a commercial kit (NucleoSpin RNA II) performed according to the manufacturer's protocol. The quality and quantity of the isolated RNA were measured with a NanoDrop ND-1000 spectrophotometer (Thermo Scientific). The RNA isolated with the QIAzol method was tested for RNA integrity with a 2100 Bioanalyzer (Agilent Technologies) and the Agilent RNA 6000 Nano Kit. The RNA isolated with the NucleoSpin RNA II kit was analyzed with the Agilent Bioanalyzer because of the low yields obtained with this method.

Reverse transcription of the isolated RNA was performed with the iScript cDNA Synthesis Kit (Bio-Rad Laboratories) according to the manufacturer's instructions. The quality and quantity of the isolated RNA was confirmed with 2 housekeeping genes, ACTB [3] (actin, beta) and HTN3 (histatin 3). Primers were designed with PRIMER3 software (http://www.genome.wi.mit. edu). Primer sequences were as follows: HTN3,5'-AATCACGGGGCATGATTATGGAGGTT-3' and 5'CTTCAAAAAGCAGTGGTAGTGTGATGC-3'; ACTB, 5'-CACCATTGGCAATGAGCGGTTC-3' and 5' AGGTCTTTGCGGATGTCCACGT-3'. cDNA (20 ng) was used as a template for all PCR amplifications. The real-time PCR was performed with a Bio-Rad thermocycler. Reactions were performed in triplicate. The PCR protocol was 95 [degrees]C for 30 s (initial denaturation) and 40 cycles of 95 [degrees]C for 2 s and 60 [degrees]C for 20 s. FaDu and Cal 27 head and neck cancer cell lines were used as positive controls for gene expression analysis. These cell lines were kindly provided by Dr. Glen Boyle from the Queensland Institute for Medical Research, Australia.

The uniqueness of the QIAzol method developed in-house is the ability to extract high yields of total RNA (0.89-7.1 [micro]g, absorbance ratio at 260/280 nm between 1.6 and 1.9) from 200 [micro]L saliva (from both the cell-free supernatant and the cell pellet) after DNase treatment. The method is cost-effective and robust, and it requires no pretreatment, such as the addition of RNase inhibitors to saliva samples. In addition, we were able to isolate RNA from archived, untreated saliva samples.

To our knowledge, this study is the first time that high yields of high-quality RNA have been isolated from both the cell-free salivary supernatant and the cellular pellet. The pellets analyzed with the QIAzol method produced relatively consistent expression profiles for the target genes, compared with the salivary supernatant (Table 1). The commercial kit produced RNA yields 10-fold lower than obtained with the QIAzol method. To address whether the QIAzol method selectively affects transcripts with different abundances, we also evaluated gene expression with the ACTB gene, which is commonly monitored as a housekeeping gene (Table 1). The peak at 25 nucleotides (nt) [4] in Fig. 1 represents an internal standard. RNA isolated from cell-free salivary supernatant with the QIAzol method yielded 5S RNA (200 nt) (Fig. 1). As expected, the FaDu and Cal 27 cell lines showed 28S and 18S bands, but salivary supernatants did not show bands of 28S (approximately 7000 nt) and 18S (approximately 4000 nt) bands. Also as expected, HTN3 (saliva-specific gene) expression was absent in the cancer cell lines. The ribosomal 18S and 28S RNAs are a part of an algorithm for calculating RNA integrity number (RIN) values, which can be used as a measure of RNA quality (RIN = 1, totally degraded; RIN = 10, intact). RNA isolated with the QIAzol method had RIN values of 2, consistent with previous findings (9).

The melting curves of the 2 housekeeping genes ACTB and HTN3 showed a single peak with similar melting temperatures (85 [degrees]C), indicating no DNA contamination and no primer-dimer artifacts. In addition, our results with the QIAzol method demonstrated that saliva does not need to be collected under RNase-free conditions and does not require the addition of an extra RNA stabilizer.

In summary, we have developed a robust, efficient, and cost-effective method for isolating high yields of RNA from saliva. We successfully isolated high yields of RNA from the cellular pellet, showing that it is possible to interrogate not only the salivary cell-free RNA but also RNA from the cellular pellet. The high-yield and good-quality RNA provided by our in-house-developed QIAzol method is useful for downstream expression analysis studies.

Previously published online at DOI: 10.1373/clinchem.2012.197863

Author Contributions: All authors confirmed they have contributed to the intellectual content of this paper and have met the following 3 requirements: (a) significant contributions to the conception and design, acquisition of data, or analysis and interpretation of data; (b) drafting or revising the article for intellectual content; and(c) final approval of the published article.

Authors' Disclosures or Potential Conflicts of Interest: Upon manuscript submission, all authors completed the author disclosure form. Disclosures and/or potential conflicts of interest.

Employment or Leadership: None declared.

Consultant or Advisory Role: None declared.

Stock Ownership: None declared.

Honoraria: None declared.

Research Funding: J. Cooper-White, Collaborative Industry Engagement Fund from the University of Queensland (UQCIEF); C. Punyadeera, Collaborative Industry Engagement Fund from the University of Queensland (UQCIEF), University of Queensland Research Foundation Excellence Award, University of Queensland New Staff Research Funds (UQNSRSF 601252), and Queensland Government Smart Futures Fellowship Programme (QGSFF).

Expert Testimony: C. Punyadeera, brain behavior and immunity.

Patents: None declared.

Role of sponsor: The funding organizations played no role in the design of study, choice of enrolled patients, review and interpretation of data, or preparation or approval of manuscript.

Acknowledgments: The authors acknowledge all the staff at the head and neck cancer clinic at the Princess Alexandra Hospital in Woolloongabba and extend special thanks to Professor William B. Coman, Associate Professor Chris Perry, and Associate Professor Ben Panizza for their continuing clinical support.

References

(1.) Pfaffe T, Cooper-White J, Beyerlein P, Kostner K, Punyadeera C. Diagnostic potential of saliva: current state and future applications. Clin Chem 2011;57:675-87.

(2.) Schulz BL, Cooper-White J, Punyadeera CK. Saliva proteome research: current status and future outlook. Crit Rev Biotechnol [Epub ahead of print 2012 May 21].

(3.) Punyadeera C, Dimeski G, Kostner K, Beyerlein P, Cooper-White J. One-step homogeneous C-reactive protein assay for saliva. J Immunol Methods 2011;373:19-25.

(4.) Tiwari M. Science behind human saliva. J Nat Sci Biol Med 2011;2:53-8.

(5.) Punnoose EA, Atwal SK, Spoerke JM, Savage H, Pandita A, Yeh RF, et al. Molecular biomarker analyses using circulating tumor cells. PLoS One 2010;5:e12517.

(6.) Debey-Pascher S, Eggle D, Schultze JL. RNA stabilization of peripheral blood and profiling by bead chip analysis. Methods Mol Biol 2009;496: 175-210.

(7.) Li Y, St. John MA, Zhou X, Kim Y, Sinha U, Jordan RC, et al. Salivary transcriptome diagnostics for oral cancer detection. Clin Cancer Res 2004;10: 8442-50.

(8.) Kumar SV, Hurteau GJ, Spivack SD. Validity of messenger RNA expression analyses of human saliva. Clin Cancer Res 2006;12:5033-9.

(9.) Wong DT. Salivary transcriptome. Clin Cancer Res 2007;13:1350-1; author reply 1351.

(10.) Lee YH, Zhou H, Reiss JK, Yan X, Zhang L, Chia D, Wong DT. Direct saliva transcriptome analysis. Clin Chem 2011;57:1295-302.

(11.) Topkas E, Keith P, Dimeski G, Cooper-White J, Punyadeera C. Evaluation of saliva collection devices for the analysis of proteins. Clin Chim Acta 2012;413:1066-70.

(12.) Dmitry A, Ovchinnikov MAC, Pratibala P, Coman WB, Cooper-White J, Patricia K, et al. Tumour-suppressor gene promoter hypermethylation in saliva of head and neck cancer patients. J Transl Oncol 2012;5:321-6.

(13.) Mohammed R, Campbell JL, Cooper-White J, Dimeski G, Punyadeera C. The impact of saliva collection and processing methods on CRP, IgE, and myoglobin immunoassays. Clin Transl Med 2012;1:19.

(14.) Foo JY, Wan Y, Kostner K, Arivalagan A, Atheron J, Cooper-White J, et al. NT-ProBNP levels in saliva and its clinical relevance to heart failure. PLoS One 2012;7:e48452.

Pratibala Pandit, [1] Justin Cooper-White, [1-2] and Chamindie Punyadeera [1,2] *

[1] Australian Institute for Bioengineering and Nanotechnology, and [2] School of Chemical Engineering, University of Queensland, St. Lucia, Queensland, Australia; * address correspondence to this author at: Saliva Translational Research Group, The University of Queensland Diamantina Institute, Level 6, TRI, 37 Kent St., Woolloongabba QLD 4102, Australia. Fax +61-(0)7-3443-6966; e-mail c.punyadeera@uq.edu.au.

[3] Human genes: ACTB, actin, beta; HTN3, histatin 3.

[4] Nonstandard abbreviations: nt, nucleotides; RIN, RNA integrity number.

Table 1. Yields of total RNA obtained with the QIAzol lysis method
developed in-house and with the NucleoSpin[R] RNA II kit
(Macherey-Nagel).

                                             Total RNA,    Absorbance
                                             ng/[micro]L   ratio,
                                                           260/280 nm

Comparison of RNA-isolation methods
       (cell-free supernatant only)
  NucleoSpin RNA II kit
    Control 1                                    1.7          0.98
    Control 2                                    1.8          0.98
    Control 3                                    1.8          1.37
  QIAzol method developed in-house
    Control 1                                   171.8         1.66
    Control 2                                   284.7         1.75
    Control 3                                   217.5         1.70
Gene expression data for RNA isolated with
       QIAzol method (c)
  Patient 1 (s)                                  326          1.93
  Patient 1 (p)                                  158          1.77
  Patient 2 (s)                                  229          1.78
  Patient 2 (p)                                  225          1.80
  Patient 3 (s)                                  209          1.75
  Patient 3 (p)                                  168          1.75
  Patient 4 (s)                                  293          1.80
  Patient 4 (p)                                  162          1.88
  Patient 5 (s)                                  256          1.80
  Patient 5 (p)                                  75           1.64
  Patient 6 (s)                                  356          1.90
  Patient 6 (p)                                  340          1.84
  Patient 7 (s)                                  171          1.82
  Patient 7 (p)                                  203          1.90
  Patient 8 (s)                                  89           1.79
  Patient 8 (p)                                  100          1.82
  FaDu cell line                                 456          2.00
  Cal 27 cell line                               577          2.20
  Control 1 (s)                                  172          1.71
  Control 1 (p)                                  158          1.66
  Control 2 (s)                                  285          1.62
  Control 2 (p)                                  124          1.58
  Control 3 (s)                                  218          1.70
  Control 3 (p)                                  224          1.73
  Control 4 (s)                                  758          1.71
  Control 4 (p)                                  987          1.76
  Control 5 (s)                                  212          1.75
  Control 5 (p)                                  824          1.78
  Control 6 (s)                                  338          1.91
  Control 6 (p)                                  209          1.72

                                              RT-PCR (a)
                                             [beta]-actin,
                                                Ct (b)

Comparison of RNA-isolation methods
       (cell-free supernatant only)
  NucleoSpin RNA II kit
    Control 1
    Control 2
    Control 3
  QIAzol method developed in-house
    Control 1
    Control 2
    Control 3
Gene expression data for RNA isolated with
       QIAzol method (c)
  Patient 1 (s)                               28.4 (0.02)
  Patient 1 (p)                               26.3 (0.05)
  Patient 2 (s)                               29.4 (0.29)
  Patient 2 (p)                               28.2 (0.02)
  Patient 3 (s)                               27.5 (0.01)
  Patient 3 (p)                               25.7 (0.02)
  Patient 4 (s)                               26.0 (0.02)
  Patient 4 (p)                               26.8 (0.04)
  Patient 5 (s)                               23.8 (0.53)
  Patient 5 (p)                               29.2 (0.02)
  Patient 6 (s)                               32.1 (0.01)
  Patient 6 (p)                               22.9 (0.06)
  Patient 7 (s)                               32.0 (0.20)
  Patient 7 (p)                               30.0 (0.63)
  Patient 8 (s)                               27.0 (0.23)
  Patient 8 (p)                               25.0 (0.15)
  FaDu cell line                              14.5 (0.09)
  Cal 27 cell line                            14.0 (0.06)
  Control 1 (s)                               27.7 (0.02)
  Control 1 (p)                               26.7 (0.23)
  Control 2 (s)                               21.7 (0.04)
  Control 2 (p)                               29.8 (0.02)
  Control 3 (s)                               23.2 (0.02)
  Control 3 (p)                               22.7 (0.04)
  Control 4 (s)                               24.4 (0.14)
  Control 4 (p)                               22.4 (0.27)
  Control 5 (s)                               19.3 (0.04)
  Control 5 (p)                               21.8 (0.23)
  Control 6 (s)                               23.8 (0.33)
  Control 6 (p)                               26.0 (0.27)

                                               RT-PCR
                                             histatin,
                                               Ct (b)

Comparison of RNA-isolation methods
       (cell-free supernatant only)
  NucleoSpin RNA II kit
    Control 1
    Control 2
    Control 3
  QIAzol method developed in-house
    Control 1
    Control 2
    Control 3
Gene expression data for RNA isolated with
       QIAzol method (c)
  Patient 1 (s)                              27.4 (0.21)
  Patient 1 (p)                              26.0 (0.02)
  Patient 2 (s)                              25.2 (0.23)
  Patient 2 (p)                              26.6 (0.21)
  Patient 3 (s)                              24.1 (0.16)
  Patient 3 (p)                              25.3 (0.03)
  Patient 4 (s)                              24.0 (0.02)
  Patient 4 (p)                              23.5 (0.22)
  Patient 5 (s)                              23.5 (0.42)
  Patient 5 (p)                              24.1 (0.09)
  Patient 6 (s)                              30.3 (0.03)
  Patient 6 (p)                              28.1 (0.01)
  Patient 7 (s)                              27.2 (0.14)
  Patient 7 (p)                              24.9 (0.07)
  Patient 8 (s)                              26.0 (0.07)
  Patient 8 (p)                              23.0 (0.06)
  FaDu cell line                             38.4 (0.01)
  Cal 27 cell line                           37.9 (0.07)
  Control 1 (s)                              24.0 (0.02)
  Control 1 (p)                              23.0 (0.09)
  Control 2 (s)                              23.9 (0.03)
  Control 2 (p)                              28.9 (0.05)
  Control 3 (s)                              19.5 (0.03)
  Control 3 (p)                              21.4 (0.02)
  Control 4 (s)                              30.8 (0.38)
  Control 4 (p)                              30.6 (0.27)
  Control 5 (s)                              26.4 (0.12)
  Control 5 (p)                              29.3 (0.07)
  Control 6 (s)                              30.6 (0.12)
  Control 6 (p)                              33.1 (0.69)

(a) RT-PCR, reverse-transcription quantitative PCR; Ct,
threshold cycle.

(b) Ct data are expressed as the mean (SD).

(c) s, Supernatant; p, pellet.
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Title Annotation:Brief Communication
Author:Pandit, Pratibala; Cooper-White, Justin; Punyadeera, Chamindie
Publication:Clinical Chemistry
Article Type:Report
Date:Jul 1, 2013
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