Steponeplus apparatus

One μg of RNA was used as template for synthesis of cDNA using the Maxima First Strand cDNA Synthesis Kit (Thermo Fisher Scientific) as per the manufacturer’s protocol. The cDNA samples were then diluted 20-fold and subjected to qPCR using the primers listed in Additional file 2. Primers were designed using the online program Primer3 (http://primer3.ut.ee) and extended across an intron when possible to eliminate the contribution from genomic DNA. qPCR was performed in triplicate using the GoTaq qPCR Mastermix kit (Promega, Madison, USA), which utilizes carboxy-X-rhodamine (CXR) as the reference fluorochrome, using the following cycling condition: 95 °C for 10 s and 60 °C for 30 s for 40 cycles. The data were collected with the StepOnePlus apparatus (Applied Biosystems, Foster City, USA) and quantitation of the samples was conducted using standard curves. LSM couples member 14B (lsm14b), prefoldin subunit 2 (pfdn2), and ring finger protein 8 (rnf8) had the most stable expression in the microarray dataset and were thus used as internal controls for qPCR. Further, 18S rRNA, beta-actin (bact), and elongation factor 1 alpha (EF1α) were also used as internal controls for qPCR [50 (link)]. The geometric means of all 6 genes were calculated and for normalization of the data quantity.

Total RNA was extracted from colonic tissues from RMT-treated mice (n = 7 per group) using TRIzol (Invitrogen, Carlsbad, CA, USA), and quantitative RT-PCR was performed as described previously [20 (link)]. In brief, total RNA was extracted and prepared for cDNA. Furthermore, quantitative RT-PCR was performed using TransStart Tip Green qPCR SuperMix (TransGen Biotech, Beijing, China) and then FastStart Universal SYBR Green Master Mix (ROX) (Roche, Switzerland, Basel) in a Step One Plus apparatus (Applied Biosystems, Foster City, CA, USA). The 2^−ΔΔCt method was utilized to determine the gene expression. GAPDH was used as an endogenous control, and all data were normalized to the control group. The primers used in this study are detailed in Additional file 1: Table S1.

Uterine tissues were treated with TRIzol (1 mL/100 mg) and RNA, was extracted as previously described (64 (link)). In brief, TRIzol-treated solution was treated with 200 μL chloroform and 500 μL isopropanol and washed with 75% DEPC water-diluted alcohol. After dissolving with DEPC water, RNA was reversely transcribed into cDNA using TransScript One-Step gDNA Removal and cNDA Synthesis SuperMix (Transgen Biotech, Beijing, China). SYBR green master (Roche, Germany) with mouse specific primer in a StepOnePlus apparatus (Applied Biosystems, Foster City, CA, USA) was performed. The oligonucleotides used are shown in Table S1. 2^-△△Ct quantification methods were used and GAPDH served as an endogenous control.

The efficiency of the subtraction method was analyzed by comparing the abundance of a nondifferentially expressed housekeeping gene (e.g., beta actin) before and after subtraction. Briefly, the subtracted and nonsubtracted samples were diluted 10-fold in H₂O as a template for PCR. Real-time PCR reactions were prepared by adding 10 μL of SYBR Premix Ex Taq (Takara, Kusatsu, Japan), 1 μL of sample, 0.8 pmol of the beta actin primers, 0.4 μL of ROX dye, and DEPC-treated water to a final volume of 20 μL. The thermal cycling parameters for the reaction consisted of an initial heating of 10 min at 95°C, followed by 40 cycles of 30 sec at 95°C and 1 min at 60°C. Melting curve analysis was performed by increasing the temperature from 65°C to 95°C in 0.1°C/second increments for each fluorescence reading using the Step-One-Plus apparatus (Applied Biosystems). The subtraction efficiency was determined using the relative expression of the beta actin gene in the subtracted and nonsubtracted samples.

Real-time PCR was used to estimate the efficiency of subtraction by comparing the abundance of a non-differentially expressed gene (a housekeeping gene: beta actin) before and after subtraction. Reactions were prepared by adding 10 μl of SYBR Premix Ex Taq (Takara, Kusatsu, Japan), 1 μl of the sample, 0.8 μl of the primers (10 μM), 0.4 μl of ROX dye and DEPC-treated water to a final volume of 20 μl. The thermal program for the reaction cycles was 10 min at 95°C, followed by 40 cycles of 30 sec at 95°C, and 1 min at 60°C. Melting curve analysis was done by increasing the temperature from 65°C to 95°C in 0.1°C/sec increments for each fluorescence acquisition, using the Step-One-Plus Apparatus (Applied Biosystems, Foster City, CA, USA). Relative expression of the beta actin gene in the subtracted and non-subtracted samples was used in the calculation of subtraction efficiency.