Dna rna shield stabilization solution

RNA was isolated from mouse tissue after homogenization in DNA/RNA Shield Stabilization Solution (Zymo, Irvine, CA) using Quick-RNA Miniprep Plus kit (Zymo) according to the manufacturer’s instructions. The resulting RNA was checked for quality using a Nanodrop. cDNA was synthesized by reverse transcription-PCR using SuperScript II Reverse Transcriptase Kit and oligo dT primers (Invitrogen, Carlsbad, CA). Real time PCR was carried out as follows: 50°C for 2 min, 95°C for 10 min, then 40 cycles of 95°C for 15 s and 60°C for 1 min with SYBR Green PCR Master Mix (Applied Biosystems, Carlsbad, CA). Gene expression was evaluated using the ΔΔCT method normalizing to the housekeeping gene m36b4 (F: 5′-ACCTCCTTCTTCTTCCAGGCTTT-3′, R: 5′-CCCACCTTGTCTCCAGTCTTT-3′) as described in (6 (link)). Genes evaluated are as follows: BAATF – 5′-TGTGATGAATAGCCCCTACCA-3′; BAATR – 5′-AGGACTGACGACTATGTCTTGTA-3′ (23 (link)), ACNAT1F - 5′-GAGGCAGCAACTGTGGTGACT-3'; ACNAT2R - 5′-TGAGACTGTATGTTTTCCTTGCTCTAC-3', ACNAT2F - 5′-AAGCGGGAACAGATTCAAGAAG-3′; ACNAT2R - 5′-ACGAAATTCAACTAGACCCCCA-3′ (24 ).

After 6 months of exposure, from 3 replicates per material, 5 circular pieces of 0.3 cm² were cut with a hole puncher and placed in cryotubes containing DNA/RNA Shield stabilization solution (Zymo Research Corporation, Tustin, CA, USA). Samples were sent to Microomics Systems S.L. (Barcelona, Spain) where they were frozen at −80 °C until analyzed. DNA was extracted using the DNeasy PowerLyzer PowerSoil Kit (Qiagen, Hilden, Germany) following the manufacturer’s instructions. The extraction tubes were agitated using Tissue lyser II (Qiagen, Hilden, Germany) at 30 Hz/s for 10 min. Mock community DNA was included as positive control for library preparation (Zymobiomics Microbial Community DNA, ZymoResearch, Irvine, CA, USA) and to ensure quality control. Samples were amplified using 16S rRNA V3-V4 regions specific primers (V3-V4-Forward 5′-TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGCCTACGGGNGGCWGCAG-3′, V3-V4 Reverse 5′GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGGACTACHVGGGTATCTAATCC-3′). Amplicon library preparation was performed as previously reported [20 (link)]. Negative controls included sample collection buffer, DNA extraction and PCR amplification blank. Sequencing was performed using an Illumina MiSeq (2 × 300 bp).

Trained healthcare workers directly observed participants as they self-collecting oral fluid and anterior nares swab specimens. Specimens placed into 10 mL collection tubes containing DNA/RNA Shield Stabilization Solution (Zymo Research, Irvine, CA). Samples were transported to the laboratory at 2–8 °C within 5 h of specimen collection and stored at 4 °C for up to 7 days before library preparation.