Step onetm real time pcr system

The participants were genotyped for the R102G C3 polymorphism (rs2230199) by a validated TaqMan SNP Genotyping Assay. SNP genotyping was performed by the Applied Biosystem Step One^TM Real-Time PCR system according to the manufacturer’s recommendations (Thermo Fisher Scientific). The assay mix (including unlabelled PCR primers, and FAM and VIC dye-labelled TaqMan MGB probes) was designed by Thermo Fisher Scientific. The reaction system utilised 20 ng of genomic DNA, 5 μl of TaqMan Universal PCR Master Mix, and 0.5 μl 20× Assay Mix and was adjusted with water for a total volume of 10 μl in each well. Alleles were scored using Applied Biosystem Step One^TM Real-Time PCR software (version 2.1).

Before qRT-PCR analysis, organoids treated with CS or/and YL20 were collected and tissue samples were harvested from mouse small intestines, followed by total RNA extraction using Trizol reagent (Invitrogen, Carlsbad, CA, USA) and RNA reverse transcription using M-MLV reverse transcriptase (Promega, Madison, WI, USA) with the primers listed in Table 1. qRT-PCR was performed with Fast SYBR Green Master Mix using the Biosystems StepOne^TM Real-time PCR system (Applied Biosystem, Waltham, MA, USA). The relative amounts of mRNA were analyzed using the 2^−ΔΔCT method.

Genomic DNA and total RNA were isolated from trophozoites using the Wizard Genomic DNA Purification kit (Promega) and Trizol reagent (Invitrogen), respectively, according to the manufacturer’s recommendations. cDNA was synthesized using oligo dT primers and the Superscript II reverse transcriptase (Invitrogen). PCR amplifications were carried out using 200 ng of DNA or cDNA as template and specific primers for Ehnpc1 or Ehnpc2a or Ehnpc2b genes (S3 Table). We used 20 μl reaction volume containing 0.5 μM each primer, 2 mM MgCl_2, 200 μM dNTPs, 1X Taq buffer and 1 U Taq DNA polymerase (Invitrogen). Cycling conditions included an initial denaturing step at 94°C for 1 min, followed by 30 cycles of 94°C for 1 min, 50 or 55°C (according to respective Tm) for 1 min, and 72°C for 3 min, with a final extension step at 72°C for 7 min. Products were separated by electrophoresis in 1% agarose gels and then, cloned and sequenced. As controls, for PCR amplification of DNA we omitted the DNA in the reaction mixture, and for RT-PCR we used DNAse-treated RNA as template. For RT-PCR assays, 100 ng of cDNA, 0.15 μM of each primer (S3 Table) and the KAPA SYBR FAST PCR Master Mix (Kapa Biosystems) were used in a StepOne^TM Real-Time PCR System (Applied Biosystem). Data from three independent cDNA preparations were analyzed using the 2^−ΔΔCt method with ribosomal 40s S2 protein as house-keeping gene.