7500ht fast real time pcr system

As previously reported [57 ], Q-PCR was carried out according to the manufacturer’s instructions. Using a high purity total RNA rapid extraction kit (Gibco BRL; Thermo Fisher Scientific), total RNA was isolated from mouse uterine tissue. In addition, the purity and integrity of RNA were evaluated using a NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific, Inc.). Genomic DNA was then removed at 42°C for 2 min, reverse transcribed at 37°C for 15 min, and reverse transcriptase inactivated at 85°C for 5 s to synthesize cDNA. Next, Quantitative real-time PCR was performed using a 7500HT fast real-time PCR system (ABI; Thermo Fisher Scientifc, Inc.). Next, Quantitative real-time PCR was performed using a 7500HT fast real-time PCR system (ABI; Thermo Fisher Scientifc, Inc.). Forty cycles at 95˚C for 30 sec and 60˚C for 30 sec were conducted, preceded by 1 min at 95˚C. Then use the 2^–ΔΔ Ct comparison method to calculate the mRNA level, and finally use the GAPDH mRNA expression normalization analysis. The following primers were used in reference to the previous literature [58 ]: TNFα sense, 5’-GTGGAACTGGCAGAAGAGGCA-3’ and antisense, 5’AGAGGGAGGCCATTTGGGAAC-3’; IL-1β sense, 5’-GTGTCTTTCCCGTGGACCTTC-3’ and antisense, 5’TCATCGAGCTGTAGTGC-3’.

Two hundred patients with newly diagnosed DLBCL and one hundred healthy controls were included in the study. Total serum miRNA was extracted using miRNeasy Serum/Plasma Kit (Qiagen, Valencia, CA, USA). MiR155 was measured by real-time quantitative RT-PCR using miScript reverse transcription Kit, using hsa-miR155 primer (MS00031486, Qiagen) and miScript SYBR Green PCR Kit (Qiagen). MiR39 (MS00019789, Qiagen) was used as endogenous control and DB cells for calibration. Total tissue miRNA was extracted using Trizol agent (Invitrogen, Carlsbad, CA, USA). RNU6 (MS00033740, Qiagen) was used as endogenous control and DB cells for calibration. The reactions were analyzed on 7500HT Fast Real-time PCR system (Applied Biosystem, Carlsbad, CA, USA). Real-time PCR was performed under the following conditions: 95 °C 15 min; 94 °C 15 s, 55 °C 30 s, and 70 °C 30 s (40 cycles). A relative quantification was calculated using the ^2-ΔΔCT method.

Total serum miRNA was extracted using miRNeasy Serum/Plasma Kit (Qiagen, Valencia, CA, USA). MiR21 was measured by real-time quantitative RT-PCR using miScript reverse transcription kit, hsa-miR21 primer and miScript SYBR Green PCR kit (Qiagen). MiR39 was used as endogenous control and DB cells for calibration. Total tissue miRNA was extracted using Trizol agent (Invitrogen, Carlsbad, CA, USA). RNU6 was used as endogenous control and DB cells for calibration. The reactions were analyzed on 7500HT Fast Real-time PCR system (Applied Biosystem, Carlsbad, CA, USA). A relative quantification was calculated using the ^2-ΔΔCT method.