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Effect of storage temperature and flash-freezing on salivary microbiota profiles based on 16S rRNA-targeted sequencing.

Author(s): Furuhashi H, Takayasu L, Isshi K, Hara Y, Ono S, Kato M, Sumiyama K, Suda W

Publication: Eur J Oral Sci, 2022, Vol. 130, Page e12852

PubMed ID: 35049092 PubMed Review Paper? No

Purpose of Paper

Conclusion of Paper

Studies

1. Study Purpose

   This study used 16S rRNA sequencing to compare microbial profiles in fresh saliva from healthy volunteers with matched aliquots that were either flash frozen in liquid nitrogen and stored at -80°C, directly stored at -80°C, or directly stored at -15°C. Unstimulated saliva was collected from five healthy volunteers (2 females, and 3 males) at least 2 h after eating. Each saliva specimen was immediately homogenized and split into four aliquots. Aliquots were: (1) immediately used for DNA/RNA extraction, (2) flash frozen in liquid nitrogen and then stored at -80°C for 14 days, (3) frozen and stored at -80°C for 14 days, and (4) frozen and stored at -15°C for 14 days. RNA was extracted using a phenol-chloroform based method and the V1–V2 region of 16S rRNA was amplified by PCR and sequenced on a MiSeq Reagent Kit v3.

   Summary of Findings:

   DNA yields of saliva specimens were unaffected by the temperature of frozen storage. The relative abundance of microbial genera was very strong when fresh control saliva specimens and saliva frozen and stored at -15°C were compared (ρ=0.950-0.988, P<0.001). Importantly, the median intraindividual correlation between specimens stored at -15°C and fresh control specimens was higher than the median interindividual correlation of fresh control specimens (ρ=0.976 versus ρ=0.603, P<0.001). Further, regression correlations were very strong between fresh control specimens and those stored at -15°C (ρ=0.976), flash frozen and stored at -80°C (ρ=0.973), and those frozen and stored at -80°C (ρ=0.983).  The α-diversity indices for the operational taxonomic units, the Chao1 index, the abundance-based coverage estimator (ACE), and the Shannon index were all comparable between fresh control specimens and each of the storage conditions. Further, the relative abundance of the 5 phylla, 20 major genera and 30 major operational taxonomic units (OTUs) levels were unaffected by the temperature of frozen storage. The weighted and unweighted UniFrac principal component analysis distance of microbial profiles did not differ among groups, and specimens clustered by patient. The median interindividual weighted and unweighted distance between fresh control and specimens stored at -15°C were smaller than the median interindividual distance of fresh control specimens (P=0.002 and P=0.005).

   Biospecimens

   • Bodily Fluid - Saliva

   Preservative Types

   • Frozen
   • None (Fresh)

   Diagnoses:

   • Normal

   Platform:

   Analyte	Technology Platform
   DNA	Next generation sequencing

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Biospecimen Preservation	Cooling or freezing method/ rate	At -80 degrees C Liquid nitrogen At -15 degrees C
   Storage	Storage temperature	-80°C -15°C
   Biospecimen Preservation	Type of fixation/preservation	Frozen None (fresh)

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