7500 ht real time pcr machine

RNA was quantified using Nanodrop spectrophotometer (Nanodrop Technologies Inc). 10 ng of the total RNA was reversely transcribed using TaqMan^® microRNA reverse transcription kit (Applied Biosystems) according to the manufactures protocol. miR-126, miR-222, miR-let7d, miR-21, miR-30, miR-92a, miR-139, miR-199a and miR-26a in circulating MPs were detected by using TaqMan^® microRNA assays (Applied Biosystems) on a 7500 HT Real-Time PCR machine (Applied Biosystems). Cel-miR-39 was used as an endogenous control. For all miRs, a Ct value above 40 was defined as undetectable. Delta Ct method was used to quantify relative microRNA expression. Values were normalized to cel-miR-39 and are expressed as 2^−ddct log 10. For all PCR experiments, samples were run in triplicates.

RNA was quantified using a Nanodrop spectrophotometer (Nanodrop Technologies Inc). Ten nanogram of the total RNA was reversely transcribed using a TaqMan microRNA reverse transcription kit (Applied Biosystems) according to the manufacturer's protocol. miRNA‐21, miRNA‐26, miRNA‐27a, miRNA‐92a, miRNA‐126‐3p, miRNA‐133a, miRNA‐222, miRNA‐223, and miRNA‐199‐5 were detected using TaqMan microRNA assays (Applied Biosystems) on a 7500 HT Real‐Time PCR machine (Applied Biosystems). Cel‐miR‐39 was used as an endogenous control. For all miRs, a Ct value >40 was defined as undetectable. Delta Ct method was used to quantify relative miRNA expression. Values were normalized to cel‐miR‐39 and are expressed as 2^−dct log 10. For all polymerase chain reaction experiments, samples were run in triplicates. The cel‐miR‐39 measurements across samples were stable and did not exceed 1 Ct value in >99% of all samples.

Total RNA was isolated out of HCAECs, monocytes, platelets, VSMCs, and EMPs by Trizol (Invitrogen) extraction method according to the instruction of the manufacturer. To increase the yield of small RNAs, the RNA is precipitated in ethanol at 20°C overnight with glycogen (Invitrogen). RNA is quantified using Nanodrop spectrophotometer. Then, 10 ng of the total RNA was reverse transcribed using TaqMan microRNA reverse transcription kit (Applied Biosystems, Life technologies, Darmstadt, Germany) according to the manufacturer's protocol. Taqman miRNA assays (Applied Biosystems) were used to measure miR-126 and miR-21 levels on a 7500 HT Real-Time PCR machine (Applied Biosystems). RNU-6 was used as an endogenous control. Delta Ct method was used to quantify relative miRNA expression.

Total mRNA was extracted using the RNAeasy mini kit (Qiagen, Crawley, U.K.) according to the manufacturer’s protocol. Briefly, EC were collected from the Transwell coculture filters using Accutase, lysed, and then added to a column. After three washes, mRNA was eluted from the column with water. mRNA concentration was measured using a NanoDrop spectrofluorometer (LabTech) and mRNA was stored at −80°C. To convert mRNA to cDNA, random primers (Promega) were annealed to 1 μg of mRNA for 5 min at 70°C, after which the following mastermix was added to give a final volume of 30 μl: 10 U of SuperScript II reverse transcriptase, 10 U of RNaseOUT, 1× SuperScript buffer (all from Invitrogen) and 10 mM dNTPs (Promega). The reaction was run at 37°C for 1 h, followed by 5 min at 95°C. To analyze mRNA, FAM-labeled E-selectin and suppressor of cytokine signaling (SOCS)3 primers and VIC-labeled 18S primers were bought as Assays-on-Demand kits from Applied Biosystems (Warrington, U.K.). Samples were amplified in duplicates using the 7500HT real-time PCR machine (Applied Biosystems) and analyzed using the software package SDS 2.2 (Applied Biosystems). Data were expressed as relative expression units relative to 18S or as fold change (2^−ΔΔCt method).

RNA was quantified using Nanodrop spectrophotometer. Exactly, 10 ng of the total RNA was reversely transcribed using TaqMan® microRNA reverse transcription kit (Applied Biosystems) according to the manufacturer’s protocol. miR-222 in plasma was detected by using Taqman miRs assays (Applied Biosystems) on a 7500 HT Real-Time PCR machine (Applied Biosystems). Cel-miR-39 was used as an endogenous control. For all miRs, a Ct value above 40 was defined as undetectable. 2^−ddCT method was used to quantify relative microRNA expression. Values were normalized to cel-miR-39.