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All samples were dried at -66C on a freeze dryer until 2 consecutive weights were obtained using 2 decimal places. Lyophilized samples were stored at -80°C until further analysis.

Approximately 100 mg of lyophilized fecal sample was pulverized, added to 15 ml Pyrex glass screw top test tubes and suspended in 1 ml of ethylene glycol-KOH. Teflon lined lids were used to seal the tubes, which were then heated on a dry heat bath for 2 hrs at 115C. Once cooled, 1 ml of aqueous NaCl was added to the tube and vortexed for 10 sec. From here 200 ml of concentrated HCL was added and samples were vortexed for another 10 sec. Tubes were allowed to cool after acidification and 6 ml of diethyl ether was added and samples vortexed for 1 min. All samples were centrifuged at 2000 g for 4 min at 4C.

The diethyl ether phase was aspirated and placed into a new tube. This extraction was repeated twice more with 6 ml of diethyl ether added each time. All extracts were pooled together and evaporated under nitrogen gas in a water bath set at 45C. The remaining residue was suspended in 3.0 ml of methanol, capped and stored at -20C until used for analysis. All reagents were brought to room temperature before analyses. To a 96-well co-star plate, 150 ml of reconstituted R3 (diaphorase, NAD+, NBT, oxamic acid) and 20 ml of sample or standard were added to the wells. Methanol was run as the blank. The plate was then incubated at 37C for 4 min. After this incubation, 30 ml of R2 (3HSD, tris buffer) was added and the plate was read immediately at 540 nm and this value was A1. After 5 min incubation the plate was read again at 540 nm to calculate A2. The absorbance of the standard and sample was calculated by subtracting A1 from A2. The concentration of the total fecal bile acid was calculated using the formula:

Fecal bile acid concentrations (mmol/L) = δ A540sample/δ A540standard standard (35 mmole/L) (4)

Values obtained from equation (4) were then converted to mmol/g of dry feces.

Fecal microbial composition analysis

DNA extraction

Stool samples were thawed at 32C for 15 min and resuspended in phosphate buffered saline (PBS) in new sterile tubes. Then, approximately 150 mg of wet mass was washed in 1 ml of PBS and centrifuged at 10,000 g for 2 min. The washing step was repeated twice. DNA was extracted from the pellets by using ZR Fecal DNA Kit (D6010, Zymo Research Corp., Orange, CA), which included a bead-beating step for the mechanical lysis of the microbial cells. We followed the manufactures' instruction except that we increased the bead-beating step to 3 min. DNA concentration and purity were determined spectrophotometrically by measuring the OD and A260/280 (Beckman DU/800, Beckman Coulter Inc., Fullerton, CA).

Primers and Real-time PCR.

Primers were assembled from the literature or newly designed and tested for specificity in silico. Those primers that did not meet our selection criteria for specificity and performance were redesigned from sequence alignments. The oligonucleotides were synthesized by University Core DNA Services (University of Calgary, Calgary, AB).

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