Pectin Impact on Bacterial Transcriptome

Total RNA was isolated from cells grown in TSB without pectin and in TSB supplemented with 0.4% w/v of either PGA, apple pectin, or RG-I from soy at 12.75 h after inoculation with the ZR Fungal/Bacterial RNA Miniprep kit (Zymo Research, Irvine, CA). Ten µg of RNA was treated with DNase using the TURBO DNA-free Kit (Thermo Fisher, Waltham, MA). RNA was used as a PCR template to confirm that no genomic DNA was remaining. One microgram of each sample was reverse transcribed with the iScript cDNA Synthesis Kit (Bio-Rad, Hercules, CA) and diluted 1:10 with nuclease-free water. Two microliter of diluted cDNA was used as template in 20-µL qPCRs with Luna Universal qPCR Master Mix (New England Biolabs, Ipswich, MA) and run on a StepOne Plus instrument (Applied Biosystems, Foster City, CA). All conditions were repeated with four biological replicates. Oligonucleotide primers were purchased from Integrated DNA Technologies (Coralville, IA).
Six potential reference genes (ftsZ, rpoD, era, adk, gyrA, and gap homologues) were evaluated and ranked using the geNorm algorithm [45 (link)] and two most stable genes, ftsZ and rpoD, were selected as endogenous control genes. Quantification thresholds and Cq values were calculated by the StepOne Plus software. Fold change in expression was calculated using the 2^−∆∆Cq method [28 (link)].