Trizol

Gene expressions of Egr-1, fibronectin (FN), E-cadherin, and α-smooth muscle (α-SMA) were analyzed by real-time quantitative PCR (RT-PCR) and performed as described previously. Total RNA was isolated from NRK-52E cells with TRIzol (Dingguo, Beijing, China). We detected the quality and concentration of the RNA by using the NanoDrop ND-1000 spectrophotometer (Thermo Fisher Scientific Inc., MA, USA). Reverse transcription of RNA was performed according to the instructions of PrimeScript RT Master Mix, purchased from Invitrogen (Carlsbad, CA, USA). RT-PCRs were proceeded by an ABI Prism 7500 real-time PCR system (Applied Biosystems, Foster City, CA, USA). SYBR Green RT-PCR MasterMix was used for quantifying the relative abundance of target mRNA. Primers are shown in Table 1. PCR reaction conditions were as follows: 95°C for 10 min, 40 cycles at 95°C for 10 s, 60°C for 20 s, and 72°C for 34 s. The relative expressions for the genes that were mentioned above were normalized to the expression of β-actin. All RT-PCRs were performed at least three separate times in triplicate and the data were presented as mean ± standard deviation (SD).

Total RNA was extracted from renal cortices and HK2 cells with TRIzol (Dingguo, Beijing, China) according to the manufacturer's instructions. After reverse-transcription using M-MLV reverse transcriptase (Invitrogen, Carlsbad, CA, USA), the gene expression levels were determined by a Roche 480 thermal cycler using 40 ng of cDNA, SYBR Select Master Mix (Invitrogen), and the respective primers (Invitrogen, Table 1). The cycling conditions were described previously [12 (link)]. The relative mRNA expression level of each gene was calculated by the comparative 2^−ΔΔCt method, with β-actin as the reference.

Total RNA was extracted using Trizol (DingGuo, China) according to the manufacturer’s instructions and the mRNA was isolated using PolyA Ttract® mRNA Isolation System III (Z5300) Kit (Promega, USA) as described in the user manual. The quality and quantity of total RNA and mRNA were assessed using a 1% sepharose gel.
A “forward” subtractive library was constructed using the PCR-Select™ cDNA subtraction kit (Clontech, USA). The mRNA isolated from HUVECs and HCMV-infected HUVECs (24 h) were designated as “tester” and “driver”, respectively. The final PCR products were purified using PCR Product Recovery kit (DingGuo) and then cloned into the pMD19-T vector (TaKaRa, China), which was then transformed into Escherichia coli DH5a cells. Transformed cells were plated onto standard LB/ampicillin/X-gal/IPTG plates at 37°C for blue/white screening. A certain number of the white colonies were taken for subsequent analysis of the sequence. Colony PCR was employed to amplify the inserted cDNA fragments with pMD19-T vector universal primers M13-47 and M13-48 (M13-47:5-CGCCAGGGTTTTCCCAGTCACGAC-3, M13-48:5-AGCGGATAACAATTTCACACAGGA-3).