Ribopure kit

From rat skeletal muscle, total RNA was extracted from the EDL and Soleus using the RiboPure kit (Applied Biosystems, Cat#AM1924, Foster City, CA, USA) according to the manufacturer’s instructions. RNA concentrations were determined in duplo by spectroscopy (ND-1000 spectrophotometer; Nanodrop Technologies, Wilmington, DE, USA). RNA purity was ensured by 260/280 ratio (range 2.00–2.11, mean 2.04). The muscle total RNA concentration was calculated on the basis of total RNA yield (μg) per weight (mg) of the analyzed sample.
For C2C12 cells, after washing cells with phosphate-buffered saline (PBS), cells were lysed in TRI reagent (Invitrogen, Cat#11312940, Carlsbad, CA, USA) and stored at −80 °C. RNA was isolated using RiboPure kit and converted to cDNA with high-capacity RNA to cDNA master mix (Applied Biosystems, Cat#4388950, Foster City, CA, USA). cDNA was diluted 10 times and stored at −20 °C.

All RNA was isolated from biofilm (three days). The S. Mutans RNA were isolated and purified by using the Ribopure Kit (Life Technology, Grand Island, NY, USA). A NanoPhotometer P360 (Implen, Westlake Village, CA, USA) was used to quantify the total RNA extracted. Reverse transcription of the RNA into cDNA was carried out by using iScript Advanced cDNA synthesis Kit for RT-qPCR (Biorad, Hercules, CA, USA) according to the manufacturer’s instructions. Real-time PCR was conducted by using iQ SYBR Green Supermix (Biorad, Hercules, CA, USA) (Klein et al., 2010 (link)). The S. Mutans primers for the genes: Glucosyltransferase (gtfB), Glucan-binding protein (gbp), at 10 μM were used. The standard curves were used to transform the critical threshold cycle (Ct) values to the relative number of cDNA molecules. Relative expression was calculated by normalizing each gene of interest to the S. mutans 16S rRNA gene, which is a well-established reference gene (Table 1). PCR amplification was performed by using 20 μL reaction mix per well in a 96 well plate. The reactions were conducted at 95 °C for 3 min, followed by 40 cycles of 15 s at 95 °C and 1 min at 60 °C. After PCR, the melting curve was obtained by incubating the samples at increasing increments of 0.5 °C from 55 to 95 °C (Klein et al., 2009 (link); Koo et al., 2006 (link)).

Whole spleen, PVAT, and the ileum were respectively homogenized in TRIzol reagent (Catalog No. 00571510, Thermo Fisher Scientific, Waltham, MA, USA), and total RNA was extracted using a RiboPure Kit (Catalog No. AM1924, Life Technologies, Carlsbad, CA, USA), according to the manufacturer's protocol. A total of 500 ng of RNA was reverse-transcribed to cDNA using a High Capacity RNA-to-cDNA Kit (Catalog No. 4387406, Applied Biosystems, Foster City, CA, USA), according to the manufacturer's protocol. The cDNAs were subsequently mixed with RT2 SYBR Green ROX qPCR Master mix (Catalog No. 1712516, Thermo Fisher Scientific), and RT-PCR was performed in accordance with the manufacturer's instructions. Thermal cycling and fluorescence detection were performed using an ABI7500 RT-PCR machine (Applied Biosystems), according to the manufacturer’s recommendations. The relative mRNA expression levels were determined using glyceraldehyde-3-phosphate dehydrogenase (Gapdh) as the housekeeping gene and the 2^−ΔΔCt method. The primers used for each gene are listed in S1 Table.