Taqman pcr kit

Total RNA, including miRNA, was isolated from 100 μl of plasma, using a 1-step phenol/chloroform purification protocol (Luo et al., 2013 (link)). An exogenous reference gene, plant miRNA MIR2911 was spiked into each sample to control for variability in the RNA extraction and purification procedures (Yan et al., 2015 (link)). Specially, to standardize the volumes (100 μl) of each plasma supernatant, we added 20 fmol (20 μl total volume) of a chemically synthesized miRNA duplex mimic of MIR-2911. RT-qPCR was performed using a TaqMan PCR kit and an Applied Biosystems 7300 Sequence Detection System to quantify the abundance of mature miRNAs. After the reactions, the cycle threshold (Ct) data were evaluated using the default threshold settings, and the mean Ct was determined from the triplicate PCRs. The relative levels of miRNAs were normalized to MIR-2911 and calculated using the 2^−ΔΔCt method. ▵Ct was calculated by subtracting the Ct values of MIR-2911 from the average Ct values of the target miRNAs. ▵▵Ct was then determined by subtracting the ▵Ct of the rest condition from the ▵Ct of the exercise conditions.

Quantitative real-time PCR (qRT-PCR) was performed by the TaqMan PCR Kit (Applied Biosystems, Foster City, CA) according to manufacturer's instructions using TaqMan probes PLK1, Hs00153444_m1; GAPDH, Hs02758991_g1. The levels of templates in samples were determined through relative quantitation (RQ) using comparative CT (ΔΔCT) assay configuration. The data were normalized by the GAPDH housekeeping gene detection. Data analysis was performed with Sequence Detection Systems 2.2.2 software (Applied Biosystems).

The expression level of the miRNAs selected for the validation step was evaluated in a total of 34 samples (20 radioresistant and 14 radiosensitive) using individual TaqMan MicroRNA Assays (Applied Biosystems). Each assay was conducted using the TaqMan MicroRNA Reverse Transcription kit (Applied Biosystems) according to the manufacturer’s protocols. Briefly, 10 ng of total RNA were reverse-transcribed using MultiScribe Reverse Transcriptase (Applied Biosystems) and a stem-loop primer (Applied Biosystems). The mixture was incubated at 16°C for 30 min, 42°C for 30 min and 85°C for 5 min. Quantitative RT-PCR (qRT-PCR) was performed using TaqMan PCR kit (Applied Biosystems) on a 7500 Fast Real-Time PCR System (Applied Biosystems). Three technical replicates of each sample were performed for every microRNA. To evaluate the differential expression of each microRNA between radioresistant and radiosensitive cases, the 2^−ΔΔCt method was employed [23 (link)]. Mean Ct values of U6 small nuclear RNA was used for normalization.

Quantitative real-time PCRs (qRT-PCRs) were performed with the TaqMan PCR Kit (Applied Biosystems, Foster City, CA) according to manufacturer's instructions, followed by detection with the Light Cycler 480 (Roche, Australia). The appropriate TaqMan probes for mRNA and microRNA quantification were purchased from Applied Biosystems. All reactions were performed in triplicate. Simultaneous quantification of GAPDH mRNAs and RNU44 were used as a reference for mRNA and microRNA TaqMan assay data normalization. The comparative cycle threshold (Ct) method for relative quantification of gene expression (User Bulletin #2; Applied Biosystems) was used to determine mRNA expression levels.

The total RNA of the MVs derived from 10⁸ cells was extracted using TRIzol Reagent (Life Technologies). Samples of total RNA were extracted for miRNAs Affymetrix GeneChip miRNA 3.0 array analysis (Life Technologies). Quantitative real-time PCR (qPCR) was performed using TaqMan microRNA probes (Applied Biosystems, Foster City, CA, USA) according to the manufacturer’s instructions. Briefly, 1 μg of the total RNA was reverse-transcribed to produce cDNA using Avian Myeloblastosis Virus reverse transcriptase (Takara, Dalian, China) and stem-loop RT primers (Applied Biosystems). Real-time PCR was performed using a TaqMan PCR kit and an Applied Biosystems 7300 Sequence Detection System (Applied Biosystems). All of the reactions, including the no-template controls, were run in triplicate. After the reactions, the CT values were determined using fixed-threshold settings.