Genomic DNA from faecal samples of colon was extracted with the QiagenQIAmp DNA Stool Minikit (Qiagen, Valencia, CA, USA), according to the manufacturer’s recommendations. The DNA concentration per microlitre was measured using a spectrophotometer, NanoDrop ND-1000 (NanoDrop Technologies Inc., Wilmington, EUA), and the readings were acquired at wavelengths of 260, 280 and 230 nm. The purity was estimated by the 260/280 nm ratio, which must range between 1.8 and 2.0 for nucleic acids. All samples were maintained at -80°C.
Lactobacillus spp. was quantified by RT-PCR. Relative levels of lactobacillus spp. DNA were quantified in real time, using a SYBR Green primer in an ABI Prism 7500 Sequence Detector (both from Applied Biosystems, Foster City, CA, USA). Relative levels of the housekeeping gene of all bacteria were measured. The primers used were: lactobacillus spp., 5′- AGC AGT AGG GAA TCT TCC A-3′ (sense) and 5′-CAC CGC TAC ACA TGG AG-3′ (antisense), and all bacteria, 5′-TCC TAC GGG AGG CAG CAG T-3′ (sense) and 5′-GAC TAC CAG GGT ATC TAA TCC TGT T-3′ (antisense). The results were obtained using the Sequence Detector software (Applied Biosystems) and were expressed as the relative increase using the method of 2-ΔΔCt, described by Livak and Schmittgen [44 (link)].
Lactobacillus spp. was quantified by RT-PCR. Relative levels of lactobacillus spp. DNA were quantified in real time, using a SYBR Green primer in an ABI Prism 7500 Sequence Detector (both from Applied Biosystems, Foster City, CA, USA). Relative levels of the housekeeping gene of all bacteria were measured. The primers used were: lactobacillus spp., 5′- AGC AGT AGG GAA TCT TCC A-3′ (sense) and 5′-CAC CGC TAC ACA TGG AG-3′ (antisense), and all bacteria, 5′-TCC TAC GGG AGG CAG CAG T-3′ (sense) and 5′-GAC TAC CAG GGT ATC TAA TCC TGT T-3′ (antisense). The results were obtained using the Sequence Detector software (Applied Biosystems) and were expressed as the relative increase using the method of 2-ΔΔCt, described by Livak and Schmittgen [44 (link)].