Trizo reagent

Total RNAs from tissues and cultured cells were extracted using Trizo reagent (Invitrogen, USA) according to the manufacturer’s protocol. cDNA was reversely transcribed by using PrimeScriptTM RT Master Mix (Perfect Real Time) (TaKaRa Biotechnology, China). All-in-One™ miRNA First-Strand cDNA Synthesis Kit (Genecopoeia, China) was used for miRNA reverse transcription and RT-qPCR was performed using All-in-One™ miRNA qPCR Kit (Genecopoeia, Guangzhou, China) of miR-200a (Cat#HmiRQP0298), miR-200b (Cat#HmiRQP0300), miR-200c (Cat#HmiRQP0302), miR-141(Cat#HmiRQP0184), miR-429 (Cat#HmiRQP0497) and U6 (Cat#HmiRQP9001). Real-time PCR was performed with SYBR Green detection chemistry (TaKaRa Biotechnology, China) on ABI 7500 Real-Time PCR System (Applied Biosystems). For relative quantification, 2^-ΔΔCt was calculated, with U6 RNA as a reference in miRNA analysis and GAPDH as a reference in the analysis of protein coding genes. The indicated primers were placed in Additional file 2: Table S2.

The presence of pluripotent and fibroblast markers in the cells of each compartment was determined using the quantitative real time polymerase chain reaction (qRT-PCR). Total RNA from the cells of each compartment of the UC was extracted using TRIzo reagent (Invitrogen) and its quality and quantity measured using a Nanodrop spectrophotometer (Nanodrop technologies, Wilmington, DW). All RNA samples were treated with DNase-I prior to first strand cDNA synthesis with random hexamers using the SuperScript first strand synthesis system (Invitrogen). Primer sequences were taken from earlier published studies and are summarized in Table 1. qRT-PCR analysis was performed with the ABI 7500 Fast Real-Time PCR System (Applied Biosystems, Foster City, CA) using SYBR green as previously described [34 (link)] and relative quantification was performed using the comparative CT (2-ΔΔCT) method.

RNA was extracted with TRIzo Reagent (Invitrogen, Germany) following the guideline provided by the manufacturer. Traces of DNA in the RNA samples were digested with TURBO Dnase (Thermo Fisher Scientific, Germany). cDNA synthesis was performed with Omniscript RT Kit (Qiagen, Germany), following manufacturer’s instructions.
Each 20 μL qPCR reaction contained 2 μL of 10× DreamTaq Buffer (Thermo Fisher Scientific, Germany), 0.2 mM dNTP, 0.5 μM forward and reverse primers, 40 ng cDNA, 1 μL 20 × Evagreen (Biotum, Germany) and 1.5 U of DreamTaq DNA Polymerase (Thermo Fisher Scientific, Germany). Real-time PCR reaction was conducted with CFX Connect Real-Time PCR Detection System (Bio-Rad, Germany). The initial denaturation step was set at 95 °C for 3 min, followed by 40 cycles of denaturation at 95 °C for 10 s, annealing at 60 °C for 50 s, and extension at 72 °C for 1 min. Melt curve analysis was performed by incubating at 95 °C for 10 s, 65 °C 5 s, and increase to 95 °C at 0.5 °C/5 s increment. Melt curve analysis showed a single peak for all genes analyzed. Values were normalized to the housekeeping gene RPS18B (AT1G34030) for gene expression analysis. Gene-specific primer pairs used in this study and the gene accession numbers are listed in Table S1.