Transcriptor first strand cdna synthesis kit

cDNA synthesis was carried out with the use of the Transcriptor First Strand cDNA Synthesis Kit (Roche Diagnostics GmbH, Mannheim, Germany) following the manufacturer’s protocol. Before cDNA synthesis, the RNA concentration of each sample was adjusted and equalized to the lowest concentration obtained during isolation (45.9 ng/μL). cDNA synthesis (total volume: 20 µL) was carried out with the use of the Transcriptor First Strand cDNA Synthesis Kit (Roche Diagnostics GmbH, Germany) following the manufacturer’s protocol in a PTC-200 thermocycler (MJ Research Inc., Saint-Bruno-de-Montarville, QC, Canada).

Total RNA from PBMCs of patients and THP-1 cells was extracted using TRIzol reagent (Invitrogen, Carlsbad, USA) and converted to complementary DNA by a Transcriptor First Strand cDNA Synthesis Kit (Roche, Basel, Switzerland). qRT-PCR was performed with a Transcriptor First Strand cDNA Synthesis Kit (Roche, Basel, Switzerland). The settings were as follows: 40 cycles of 10 s at 95 °C, 30 s at 60 °C, and 30 s at 72 °C. All relative mRNA expression levels were analyzed using the 2^−ΔΔCt method. The primers used are listed in Table S1.

Total RNA was extracted from cells using TRIzol method. The extracted RNA was reversely transcribed with a Transcriptor First Strand cDNA Synthesis Kit (Roche Diagnostics GmbH Mannheim, Germany). The PCR was performed using a Real-Time PCR System (Illumina, USA) under the following conditions: 95°C for 10 min, followed by 40 cycles of 95°C for 10 s, 55°C for 30s, and 72°C for 60 s. Results were analyzed with the 2^-ΔΔCt method. The mRNA levels were normalized to GAPDH. The primer sequences for Simian RV VP6 [GenBank: L15384.1] were designed as 5’-CTTCTACCAGACGCGGAAAG-3’ (forward) and 5’-ATTCGGCCTGAGAATCACTG-3’ (reverse).
Total RNA was extracted by a single-step isolation procedure using Trizol (Invitrogen, USA). Total RNA were synthesized into cDNA using the Transcriptor First Strand cDNA Synthesis Kit (Roche Diagnostics GmbH). Semi-quantitative RT–PCR was performed to determine expression levels. GAPDH was used as a loading control. All primers were synthesized by SANGON (China). The sequences of the primers used were listed as follows: phosphatidylinositol 3-kinase (PI3K): Forward, 5’-CATCACTTCCTCCT GCTCTAT-3’, Reverse, 5’-CAGTTGTTGGCAATCTTCTTC-3’; protein kinase B (AKT), Forward: 5’-GGACAACCGCCATCCAGACT-3’, Reverse: 5’-GCCAGGGACACCTCCATCTC-3’; GAPDH: Forward, 5’-GGAGCGAGATCCCTCCAAAAT-3’, Reverse, 5’-GGCTGTTGTCATACTTCTCATGG-3’.

RNAsimple (Tiangen, Beijing, China) was used to isolate total RNA from heart tissues and H9c2 cells. Using the Transcriptor First Strand cDNA Synthesis Kit (Roche, Basel, Switzerland), the RNA was reverse transcribed into cDNA. The RT procedure was 37 ℃ for 15 min and 85 ℃ for 5 s. After that, SYBR Green PCR Kit (Takara, Dalian, China) was used for qRT-PCR in a PCR system. The amplification process was as follows: denaturation at 95 ℃ for 5 min, followed by 40 cycles at 95 ℃ for 15 s, annealing at 55 ℃ for 30 s, and extension at 60 ℃ for 60 s. GAPDH or U6 was used as the internal control. Data were analyzed using the 2^−ΔΔCt method. The primer sequences were as follows: circRbms1, F 5′-CTGAGCCTGGACTCCATTCG-3′, R 5′-ACCAGGAGTTTCTGGTTATGGT-3′; Rbms1, F 5′-CTGAGCAAGACAAACCTCTACAT-3′, R 5′-GGCCTTATCCAAAATCGCCTT-3′; miR-742-3p, F 5′-GCCGAGGAAAGCCACCATGCTGG-3′, R 5′-CAGTGCGTGTCGTGGAGT-3′; FOXO1, F 5′-CCCAGGCCGGAGTTTAACC-3′, R 5′-GTTGCTCATAAAGTCGGTGCT-3′; GAPDH, F 5′-GGTGAAGGTCGGTGTGAACG-3′, R 5′-CTCGCTCCTGGAAGATGGTG-3′; U6, F 5′-CTCGCTTCGGCAGCACATATACT-3′, R 5′-ACGCTTCACGAATTTGCGTGTC-3′.

cDNA was synthesized using Transcriptor First Strand cDNA Synthesis Kit (Roche, Germany) by reverse transcription according to the manufacturer’s instructions. Reverse transcription was carried out in 20 μl solution that contained 0.5 μl 20 U/μl Transcriptor reverse transcriptase, 4μl Transcriptor RT Reaction Buffer (5x concentrated), 2 μl Deoxynucleotide Mix, 0.5 μl 40 U/μl protector RNase inhibitor, 2 μl 10 mmol/ml stem-loop RT primers (Invitrogen), 7 μl DEPC water (Invitrogen, USA), and 4 μl total RNA template. After being mixed gently, the reaction mixtures were incubated at 25°C for 10 min, 55°C for 30 min and then 85°C for 5 min. The final cDNA products were stored at −20°C until use. U6 and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were used as the reference genes. The sequence of all primers used in the present study is provided in Table 2.