NIH, National Cancer Institute, Division of Cancer Treatment and Diagnosis (DCTD) NIH - National Institutes of Health National Cancer Institute DCTD - Division of Cancer Treatment and Diagnosis

Stability of circulating miRNA in saliva: The influence of sample associated pre-analytical variables.

Author(s): Romani C, Baronchelli M, Assoni C, Mattavelli D, Calza S, Piazza C, Bossi P

Publication: Clin Chim Acta, 2023, Vol. , Page 117702

PubMed ID: 38097127 PubMed Review Paper? No

Purpose of Paper

This paper compared levels of microRNA (miRNA, miR)-484 and miR-106b-5p in saliva collected from healthy volunteers and patients with oral cancer by either spitting or using a LolliSponge device. The authors also compared levels of miR-484 in saliva specimens that were collected from LolliSponge devices and stored for 0, 24, 48 or 96 h before processing and freezing at -80°C and between saliva specimens that were stored for a year at -20°C rather than -80°C.

Conclusion of Paper

miR-485 quantification cycles (Cq) were comparable between saliva specimens collected by spitting and those collected with the LolliSponge device for specimens collected from volunteers and oral cancer patients. Additionally, miR-106b-5p levels that were normalized to miR-484 showed good agreement between the collection methods evaluated. While miR-484 levels were unaffected by storage of the LolliSponges at 4°C for up to 48 h before centrifugation, miR-484 levels were significantly lower in specimens that were stored for 96 h than immediately processed specimens and in specimens stored at -20°C compared to those stored at -80°C.

Studies

  1. Study Purpose

    This study compared levels of miR-484 and miR-106b-5p in saliva collected from healthy volunteers and patients with oral cancer by spitting and using a LolliSponge device. The authors also compared levels of miR-484 in saliva specimens collected from LolliSponge devices that were stored for 0, 24, 48 or 96 h before processing and freezing at -80°C and between saliva specimens that were stored for a year at -20°C rather than -80°C. Saliva was collected from 20 healthy volunteers (13 females and 7 males) and 8 patients with oral cancer (2 females and 6 males) by spitting into a Falcon tube and then using a LolliSponge device. LolliSponges were stored for 24, 48, or 96 h before processing or released from the sponge by centrifugation at 450 g for 60 seconds. Cells were separated from the saliva in Falcon tubes and released from LolliSponges by centrifugation at 2600 g for 15 min at 4°C and the cell-free supernatant was stored at -80°C until RNA isolation using the miRNeasy Mini Kit. miR-484 and miR-106b-5p were quantified using the miRCURY LNA miRNA PCR assay. To test the effects of saliva storage temperature, case-matched saliva supernatant was stored for 1 year at -80°C and -20°C until RNA isolation.

    Summary of Findings:

    Cq values for miR-484 were comparable between saliva specimens that were collected by spitting or using the LolliSponge device in both volunteers and oral cancer patients. miR-484 Cq values showed a linear relationship with RNA input, indicating that neither collection method resulted in PCR inhibition. Additionally, miR-106b-5p levels that were normalized to miR-484 showed good agreement between the collection methods (bias =-0.175). miR-484 levels were unaffected by storage of the LolliSponges at 4°C for up to 48 h before centrifugation but were significantly lower in specimens stored for 96 h compared to immediately processed specimens (P=0.0032). miR-484 levels were lower when saliva was stored at -20°C rather than -80°C (P<0.001). 

    Biospecimens
    Preservative Types
    • Frozen
    Diagnoses:
    • Neoplastic - Carcinoma
    • Normal
    Platform:
    AnalyteTechnology Platform
    RNA Real-time qRT-PCR
    Pre-analytical Factors:
    ClassificationPre-analytical FactorValue(s)
    Biospecimen Acquisition Method of fluid acquisition Spitting
    LolliSponge
    Preaquisition Diagnosis/ patient condition Healthy
    Oral cancer
    Storage Storage temperature -20°C (1 year)
    -80°C (1 year)
    Real-time qRT-PCR Specific Targeted nucleic acid miR-484
    miR-106b-5p
    Storage Storage duration 0 h (at 4°C)
    24 h (at 4°C)
    48 h (at 4°C)
    96 h (at 4°C)

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