Geneamp pcr system 2400 thermal cycler

RNA was isolated by using a NucleoSpin RNA II kit (Macherey-Nagel GmbH) and cDNA prepared with a Reverse Transcription System kit (Promega Corp). Quantitative real-time polymerase chain reaction was performed using predesigned Taqman Gene Expression Assays and AmpliTaq Gold DNA polymerase following the manufacturer's instructions (Applied Biosystems Inc.). The ratio of the mRNA levels for each sample was calculated by normalizing the comparative quantitation values to those of GAPDH mRNA. PCR was performed in a GeneAmp PCR system 2400 Thermal Cycler (Perkin-Elmer, Norwalk, CT, USA). Primers used were as follows: GAPDH, 5′-ACG GCA AAT TCA ACG GCA CAG TCA-3′ (forward) and 5′-TGG GGG CAT CGG CAG AAG G-3′ (reverse); Collagen I, 5′-TGC CGT GAC CTC AAG ATG TG-3′ (forward) and 5′-CAC AAG CGT GCT GTA GGT GA-3′ (reverse); Collagen III, 5′-AGA TCA TGT CTT CAC TCA AGT C-3′ (forward) and 5′-TTT ACA TTG CCA TTG GCC TAG-3′ (reverse).

The whole blood from patients were used for detection. RNA was extracted by Trizol reagent (Life Technologies, United States). ACTIN was used as the reference gene. The cDNA was then reverse-transcribed using the First Strand cDNA Synthesis Kit (TOYOBO, Japan). Then, the cDNA samples were subjected to real-time quantitative PCR using a TransStart Top Green qPCR SuperMix (TransGen Biotech, China) on a GeneAmp PCR System 2400 Thermal Cycler (PerkinElmer, Wellesley, MA, United States).

Total RNA was extracted by using TRIZOL reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's protocol. The first strand cDNA was generated from total RNA with reverse transcriptase (TAKARA, Shiga, Japan) and used as the template for qRT-PCR analysis. GAPDH cDNA was used as an internal control to normalize variances. Primers used were as follows: lin28a, 50-GAGGCAGTGGAGTTCACCTTTA-30 (forward) and 50-TCCTTGGCATGGTGGTCTA-30 (reverse); GAPDH, 50-GGCACAGTCAAGGCTGAGAATG-30 (forward) and 50-ATGGTGGTGAAGACGCCAGTA-30 (reverse); let7a, 50-CGGTGAGGTAGTAGGTTGTATAGTT-30 (forward). PCR was performed in a GeneAmp PCR system 2400 Thermal Cycler (Perkin-Elmer, Norwalk CT, USA). PCR conditions were 30 sec. at 94°C, 30 sec. at 58°C and 30 sec. at 72°C (30 cycles). The PCR products were detected by real-time PCR detection kit (RR716; TAKARA) in ABI 7500 sequence detection system.