M mlv reverse transcriptase cdna synthesis kit

TRIzol reagent (Invitrogen, Carlsbad, CA) was applied to extract total RNA from cells or patient samples. Reverse transcription was performed using M-MLV reverse transcriptase cDNA Synthesis Kit (Takara, Japan). Real-time RT-PCR was carried out on ABI 7900HT Fast Real-Time PCR System (Foster City, CA) with SYBR-Green PCR Master Mix (Toyobo, Japan). Melting curves analysis was applied to guarantee the specificity of amplification. A comparative CT method (2^−ΔΔCT) was used to analyze the gene expression level. β-actin or GAPDH was used as the internal control. The primers for real-time RT-PCR are available in Table 1.

Total RNA was isolated from the kidneys of individual mice or patients using TRIzol (TaKaRa, Dalian, China). cDNA was synthesized using the M-MLV Reverse Transcriptase cDNA Synthesis Kit (TaKaRa) according to the manufacturer’s instructions. Real-time PCR was performed with an ABI Prism 7300 Sequence Detection system (Applied Biosystems) using the SYBR^® Premix Ex Taq TM II (TaKaRa). The gene expression in each sample was analyzed in duplicate and normalized against the internal control gene GAPDH. Relative quantification of the target gene expression in patients compared with normal samples was performed with the ΔΔCt method

The quantitative real-time PCR was performed as described in other study [20 (link)]. Briefly, total RNAs were isolated from cultured BUMPT cells or kidney tissues with the TRIzol (Thermo Fisher Scientific). For qPCR analysis of miRNAs, 50 ng of total RNAs from each sample were reversely transcribed into cDNA by using the microRNA Reverse Transcription kit (Applied Biosystems), and qPCR was performed with a TaqMan miRNA assay kit (4440887; Applied Biosystems). For qPCR analysis of mRNAs, 1ug of total RNAs from each sample were reversely transcribed into cDNA by using an M-MLV Reverse Transcriptase cDNA Synthesis Kit (TaKaRa), and qPCR was performed with TB GreenTM Premix Ex Taq II reagent (TaKaRa). All PCR data were analyzed by LightCycler 96 SW 1.1 software, and relative levels were determined by the 2-ΔΔCt method. For miRNA analysis, miR-26a-5p was normalized to the level of U6 (internal control) to determine the ratios. The ratios of control mice were arbitrarily set as 1. For mRNA analysis, IL-1, IL-6 or TNF-α mRNA (Primers applied as shown in Table 1) was normalized to the level of GAPDH (internal control) to determine the ratios. The ratios of control mice were arbitrarily set as 1.

Real-time PCR reactions were performed with the ABI detection system (Applied Biosystems, Foster City, CA, USA). Reverse transcription was performed using 1 µg of total RNA from each sample and the MMLV Reverse Transcriptase cDNA Synthesis Kit (TaKaRa, Dalian, China), according to the manufacturer's instructions. RT-PCR was performed using SYBR Green (TaKaRa, Dalian, China) in the ABI 7500 Sequence Detection System. PCR primer sequences were designed for the candidate genes and the GAPDH housekeeping gene using Primer Premier 5.0 software, and their sequences are listed in table 1. Each RT-PCR reaction was performed in triplicate. For analysis, fluorescence signal values were obtained using ABI 7500 Sequence Detection System software, and the Ct value of each sample and gene was obtained. The 2^−ΔΔt method was used to calculate the differences in gene expression in multiples.
Table 1.

Primer information for each gene.

GenBank accession number	gene symbol	primer sequences (5′ → 3′)
NM_001082253.1	GAPDH	sense	GGAGAAAGCTGCTAA
		antisense	ACGACCTGGTCCTCGGTGTA
XM_002724032.2	BMP7	sense	GGGCTTCTCCTACCCCTACA
		antisense	TTGTCGTGTTCCACGAGGTT
NM_001177330.1	LPL	sense	GAAACTCAAGTGGAACAGCGAC
		antisense	TCAGAGACTTGTCGTGGCATTT
NM_001171013.1	MCP2	sense	AAGTCGTAGACCAGCAGCCC
		antisense	GCAGCAGAGTGGGTGGATTCT
XM_002716499.3	SST	sense	CCCAACCAGACGGAGAATGA
		antisense	AGGGATTCTGGGGGATTAGG