Integrating Electrical Activities of Heart and Stomach with gut microbial composition to Evaluate the Association of Gut-Heart Axis Modulation with Thermoregulation of Hyperthermic Rats

Furthermore, 5’AGAGTTTGATGMTGGCTCAG 3’ and 5’TTACCGCGGCMGCSGGCAC 3’ used as forward and reverse primers, respectively. 40ng of the purified DNA was used as the PCR amplification template with 10pM of both the primers and the triplicates were done for obtaining the best result using protocol: preheating at 95℃ for 3 minutes; 25 cycles of denaturation at 95℃ for 15 seconds, annealing at 60℃ for 15 seconds, elongation at 72℃ for 2 minutes and the final extension was carried out at 72℃ for 10 minutes then held at 4℃.

Now, the amplified 16s PCR product were subsequently used for the downstream analyses and for the quality check subjected to Nano Drop QC (Thermo Fisher Scientific, Waltham, MA, USA) reading of 260/280 at a value of approximately 1.8 to 2. The amplicons from each sample were purified with Ampure beads to remove unused primers and an additional 8 cycles of PCR was performed using Illumina barcoded adapters to prepare the sequencing libraries.