gender: male tissue: Left Mandibles treatmen: infected with Tannerella forsythia time point: 8 weeks
Treatment protocol
100 million bacterial cells suspended in reduced transport fluid is mixed with equal volume of 6% carboxymethylcellulose (CMC) and administed orally. 0.1 ml cell suspension/mice.
Growth protocol
Tannerella forsythia ATCC 43037 (Tf) was grown in tryptic soy broth supplemented with N-acetyl muramic acid (5 mg/mL) and hemin (1 mg/mL) for three days in anaerobic growth chamber.
Extracted molecule
total RNA
Extraction protocol
Total RNA isolated from the mandible tissue using mirVana miRNA isolation kit (Ambion, Austin, TX, USA). mandibles from each mouse were entirely homogenized using the handheld rotor-stator homogenizer with sterile individual TissueRuptor disposable probes (Qiagen; Germantown, MD, USA) for each sample. After homogenization, each sample was lysed in a denaturing lysis solution that stabilized RNAs and inactivated RNases. The samples were subjected to an acid-phenol:chloroform extraction that removed all the cellular components such as protein, DNA, and other cellular products. The aqueous phase was removed, transferred to the fresh nuclease-free microcentrifuge tube, and 1.25 volume of 100% ethanol was added. This mixture was transferred to the filter cartridge placed into the collection tube and centrifuged for 15 s at 10,000× g. The filter cartridge was washed with 700 µL of wash solution-1 followed by washing with wash solution-2/3. After washing, the total RNA was eluted from the filter cartridge with 100 µL of nuclease-free water. RNA yield and purity were determined using a Take3 micro-volume plate in Epoch Microplate Spectrophotometer (BioTek), and quantification was performed in technical duplicates for each sample.
Label
n.a.
Label protocol
n.a.
Hybridization protocol
We used high-throughput nCounter® miRNA Expression Panels (Nanostring Technologies, Seattle, WA, USA) to effectively identify the DE of miRNA in both sexes. Nanos- Int. J. Mol. Sci. 2022, 23, 5107 14 of 18 tring nCounter® analysis can identify 577 miRNAs in any sample, and by using molecular barcodes, it can detect even a low number of miRNAs without the need for reverse transcription or amplification. miRNA expression profiling was performed using the Nanostring nCounter® Mouse miRNA Assay kit v1.5 (Nanostring Technologies). This assay is a highly sensitive multiplexed method that detects miRNAs using molecular barcodes called nCounter reporter probes without the need for reverse transcription. Sample preparation involved annealing, ligation, and purification. These steps were carried out based on the experimental procedure described in the nCounter® miRNA assay panel kit. Briefly, the annealing master mix was prepared by combining 13 µL of annealing buffer, 26 µL of nCounter miRNA Tag reagent, and 6.5 of diluted (1:500) miRNA assay controls. Next, 3.5 µL of the annealing master mix was aliquoted into each tube of the strip tube and 100 ng of the total RNA from six left mandibles from each group (maximum 3-µL volume) with 260/280 and 260/230 ratios of greater than 2 was added to the respective tubes. The strip tube was placed in the Thermal Cycler with the following conditions: 94 ◦C for 1 min, 65 ◦C for 1 min, 45 ◦C for 1 min, and 48 ◦C for hold. After annealing, 2.5 µL of the ligation master mix (19.5 µL of polyethylene glycol (PEG) and 13 µL of ligation buffer) was added to all the tubes in the strip tube. The strip tube was incubated at 48 ◦C for 5 min, followed by the addition of 1 µL of ligase into each tube without removing the strip tubes from the Thermal Cycler. Ligation was performed with the following conditions: 48 ◦C for 3 min, 47 ◦C for 3 min, 46 ◦C for 3 min, 45 ◦C for 3 min, 65 ◦C for 10 min, and 4 ◦C for hold. To remove the unligated tags, a purification step was performed after adding 1 µL of ligation cleanup enzyme to all the tubes and incubating the tubes at 37 ◦C for 1 h, 70 ◦C for 10 min, and 4 ◦C for hold. Forty microliters of RNase-free water were added to each tube in the strip tube and the sample was ready for hybridization with the nCounter reporter and capture probes. After denaturation at 85 ◦C for 5 min, a 5-µL aliquot from the miRNA sample preparation tube was taken along with the 10 µL of miRNA reporter code, 10 µL of hybridization buffer, and 5 µL miRNA capture probe. The strip tubes were incubated at 65 ◦C for 18 h in the Thermal cycler, and the samples were immediately processed for post-hybridization with the nCounter analysis system at the Molecular Pathology Core at the University of Florida. The nCounter® Mouse miRNA Assay kit v1.5 provided six positive hybridization controls and eight negative control probes to monitor hybridization efficiency. All components and reagents needed for sample preparation at the reparation station were taken from the nCounter master kit (Nanostring Technologies). Twelve samples per cartridge were processed in a single run, which took 3 h. This was followed by digital analysis, which involved the transfer of the cartridge to the multichannel epifluorescence digital analyzer. A cartridge definition file with a maximum fields of view (FOV) count of 555 per flow cell was taken for digital analysis. The number of images taken per scan corresponded to the number of immobilized reporter probes on the cartridge. A separate Reporter Code Count (RCC) file for each sample containing the count for each probe was downloaded and used for data analysis.
Scan protocol
n.a.
Description
miRNA expression profiling was performed using the Nanostring nCounter® Mouse miRNA Assay kit v1.5 (Nanostring Technologies).
Data processing
Initial data analysis was performed using nSolver 4.0. Initially, RCC files were imported into nSolver and imaging quality control (QC), binding density QC, positive control linearity QC, and positive control limit of detection QC were carried out as recommended in the system QC parameters. The lanes were flagged when the percent FOV registration was less than 75% for imaging QC. Binding density was outside the range from 0.1 to 2.25 for binding density QC, and the positive control R2 value was less than 0.95. All 24 samples passed the QC, and no flag lanes were observed. Raw data were generated after passing the QC. To reduce the background signal/noise, the background threshold count value was set to 52 and calculated by taking an average of eight negative control probe counts from all 24 samples. Codeset content normalization parameters were chosen, and the normalization was performed based on the top 100 miRNA genes expressed. The normalized factor was calculated based on the geometric mean values of the miRNA genes expressed in each sample. Fold changes in the genes were calculated based on the ratio of the difference in the means of the log-transformed normalized data to the square root of the sum of the variances of the samples in the two groups. The limma R library was used to calculate fold changes. Clustering of miRNA for the final heatmap of DE miRNA was done using the Partitioning Around Medoids method with the fpc R library.
