Investigating the Effects of Traffic-Generated Air-Pollution on the Microbiome and Immune Responses in Lungs of Wildtype Mice Page: 26
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manufacturer's recommendations. The samples were processed in triplicates, and values were
determined from a known value standard curve, using a sigmoidal four-parameter logistic (4-PL)
curve-fit.
2.3.3 qPCR
DNA from homogenized lung tissues (n=6 per group) was extracted using ZR Fecal DNA
miniprep (Zymo Research). qPCR was performed using SsoAdvanced Universal SYBR Green
Supermix (Bio-Rad) and the CFX96 Real-Time system (Bio-Rad). For bacterial 16S rRNA
analysis, samples were normalized to Eubacteria utilizing known-concentration standards.
Bacterial primers used are described in Table 2.1.
Table 2.1: Primer sequences used for qPCR analysis.Eubacteria FP
Eubacteria RP
Enterobacteriaceae FP
Enterobacteriaceae RP
Bacteroidetes FP
Bacteroidetes RP
Firmicutes FP
Firmicutes RP
Actinobacteria FP
Actinobacteria RP5'-ACTCCTACGGGAGGCAGCAGT-3'
5'-ATTACCGCGGCTGCTGGC-3'
5'-GTGCCAGCMGCCGCGGTAA-3'
5'-GCCTCAAGGGCACAACCTCCAAG-3'
5'-GGTTCTGAGAGGAGGTCCC-3'
5'-GCTGCCTCCCGTAGGAGT-3'
5'-GGAGYATGTGGTTTAATTCGAAGCA-3'
5'-AGCTGACGACAACCATGCAC-3'
5'-CGCGGCCTATCAGCTTGTTG-3'
5'-CCGTACTCCCCAGGCGGGG-3'2.3.4 Illumina MiSeq Sequencing
Genomic DNA was isolated using ZR Fecal DNA miniprep (Zymo Research) from lung
homogenates (n=6 per group). 16S rRNA genes (variable region 4, V4) were amplified using a26
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Daniel, Sarah. Investigating the Effects of Traffic-Generated Air-Pollution on the Microbiome and Immune Responses in Lungs of Wildtype Mice, dissertation, December 2020; Denton, Texas. (https://digital.library.unt.edu/ark:/67531/metadc1752346/m1/38/?q=j+w+gardner: accessed July 6, 2024), University of North Texas Libraries, UNT Digital Library, https://digital.library.unt.edu; .