Sybr green gene expression assay

Total RNA from tumor tissues and cell lines was extracted using TRIzol reagent (Invitrogen, #15596026) according to the manufacturer’s instructions. The concentration and purity of the isolated RNA were determined using a NanoDrop spectrophotometer (Thermo Fisher Scientific). Complementary DNA (cDNA) was synthesized from the total RNA using a reverse transcription kit (Thermo Fisher Scientific, #K1622) following the manufacturer’s protocol. Quantitative real-time PCR was performed using a specific SYBR Green Gene Expression Assay (Applied Biosystems, #4309155) for the FYN gene and an endogenous control (ACTB) on a real-time PCR system. FYN: forward, 5′-CAG​TTG​ACT​CCA​TCC​AGG​CAG​A-3′, and reverse, 5′-CAC​GGA​TGG​AAA​GTG​AGT​AGG​C-3′.
PIK3R1: forward 5′-CGC​CTC​TTC​TTA​TCA​AGC​TCG​TG-3′, and reverse, 5′- GAA​GCT​GTC​GTA​ATT​CTG​CCA​GG-3′. ACTB: forward, 5′-CAT​TGC​TGA​CAG​GAT​GCA​GAA​GG-3′, and reverse, 5′-TGC​TGG​AAG​GTG​GAC​AGT​GAG​G-3′. Each reaction was run in triplicate. The relative expression levels of the FYN gene were calculated using the 2^(−ΔΔCt) method, where Ct represents the threshold cycle, and data were normalized to the endogenous control. The qPCR results presented in display the average RNA expression level of FYN and PIK3R1 across three mice.

Total RNA was extracted using Micro Scale RNAqueous Isolation kit, and then cDNA was synthesized with the High-Capacity cDNA Reverse Transcription kit (both from Applied Biosystems, Foster City, USA). Quantitative real-time polymerase chain reaction was performed with the SYBR green Gene Expression Assay (Applied Biosystems, Foster City, USA). The relative expressions of target genes were calculated using the 2^ΔC(t) method. The sequences of primers for polymerase chain reaction are as follows: intercellular adhesion molecule (ICAM)-1, 5′-CTCATCCTGCGCTGTCTGGT-3′ (forward), 5′-CCGGAGCTGCCTGACCTCGG-3′ (reverse); CXCL1, 5′-GCGTTTCATCGATGGTCGTT-3′ (forward), 5′-CTTCTTCCCGCTCAACACCT-3′ (reverse); and CXCL2, 5′-AACCATCAGGGTACAGGGGT-3′ (forward), 5′-GGGCTTCAGGGTTGAGACAA-3′ (reverse).

Total RNA was harvested from tibia, epiphysis or cultured chondrocytes using RNeasy Plus Mini Kit (Qiagen). Complementary DNA was then synthesized with iScript reverse transcription kit (Bio-Rad) and quantified using primers listed in Supplemental Table 1. Gene expression was analyzed by SYBR Green gene expression assay (Applied Biosystems).

The human uroepithelial cell line T24 was infected by co-incubation with isogenic strains of E. coli for 2 h^{99 (link)}. Total RNA was extracted using the RNeasy Mini kit (Qiagen) according to the manufacturer’s protocol. The concentration and purity of RNA were determined with nanodrop, and 300 ng of RNA was transcribed to cDNA using the High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems). Real-time PCR for human IL1B (forward 5′-CAC GAT GCA CCT GTA CGA TCA-3′, reverse 5′-GTT GCT CCA TAT CCT GTC CCT-3′), CXCL8 (forward 5′-AAG AGA GCT CTG TCT GGA CC-3′, reverse 5′-GAT ATT CTC TTG GCC CTT GG-3′) were analyzed in comparison to housekeeping gene ACTB (forward 5′- AAG AGA GGC ATC CTC ACC CT-3′, reverse 5′-TAC ATC GCT GGG GTG TTG-3′) using SYBR green gene expression assay (Applied Biosystems) in a Rotor-Gene PCR cycler (Corbett Life Science). Relative expressions of target genes was presented as 2^−∆CT and fold change as 2^−∆∆CT compared to vector control infected cells.

Total RNA was extracted using either the RNeasy Mini kit (Qiagen) or Arcturus PicoPure RNA isolation kit (Applied Biosystems), and reverse transcription was performed with the High-Capacity cDNA Reverse Transcription kit (Applied Biosystems) according to the manufacturers’ instructions. qPCR was performed with the SYBR Green gene expression assay (Applied Biosystems) using the ViiA 7 Real-Time PCR System (Applied Biosystems) and analyzed with Quant Studio Real-Time PCR software v.1.3 (Applied Biosystems). Primer pairs are listed in Supplementary Table 11.