7900ht fast real time pcr system

miTRAP cDNA for use with TaqMan miRNA multiplex arrays was first preamplified using TaqMan PreAmp Master Mix (ThermoFisher, 4,391,128) and custom miRNA PreAmp primer pool. The qPCR reactions for miRNA profiling experiments and miTRAP experiments were prepared using TaqMan Universal Master Mix II with UNG (ThermoFisher, 4,440,038), and custom miRNA microfluidic cards were run on an Applied Biosystems 7900 HT Fast Real Time PCR System fitted with the 384-well block. qPCR reactions for miR-375 were prepared using Universal Master Mix II with UNG and TaqMan small RNA assay for miR-375 (ThermoFisher, 4,427,975, 000564), and were run in MicroAmp fast 96-well reaction plates (0.1 ml, Applied Biosystems, 4,346,907) covered with optical adhesive covers (Applied Biosystems, 4,360,954) using the 7900 HT Fast Real Time PCR System fitted with the 96-well block. All protocols were performed following the manufacturer’s instructions.
qPCR reactions for TurboGFP were performed using QuantiTect SYBR Green PCR kit (204143) and run using the Stratagene Mx300P qPCR system (Agilent Genomics). PCR reaction settings were as follows: hot start for 15 min at 95 °C, and amplification 15 s at 95 °C, 30 s at 60 °C, 30 s at 72 °C repeated for 40 cycles with data recorded twice during the extension step.

Total RNA (100 ng) was reverse-transcribed to cDNA using a High Capacity cDNA Archive Kit (Life technologies; Carlsbad, CA, USA). TaqMan PCR was carried out using the TaqMan Fast Advanced PCR master mix and TaqMan gene expression assays (all reagents from Life Technologies), by means of a 7900HT Fast Real-Time PCR System (Applied Biosystems). Assays were performed in triplicate. Gene expression profiling was achieved using the comparative cycle threshold (CT) method of relative quantitation using Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as housekeeping genes. To normalize data, ΔΔCT was calculated for each sample using the mean of its ΔCT values subtracted from the mean ΔCT value measured in the control sample, set as a calibrator; relative quantitation (RQ) value was expressed as 2^−ΔΔCT.
Individual miRNA detection by RT-qPCR was performed using the TaqMan MicroRNA assays (Life technologies). Individual reverse transcription reactions (5 ng of total RNA each target) were performed using the Taqman microRNA Reverse Transcription Kit and the miRNA-specific looped-primers. TaqMan PCR was performed in triplicate by using the 7900HT Fast Real-Time PCR System (Applied Biosystems). miRNA expression RQ data were calculated as reported above, using U6 snRNA as housekeeping control.

Expression of mature miRNAs was measured by using the TaqMan® Low Density Arrays V2 containing 590 small RNAs with Applied Biosystems 7900HT Fast Real-time PCR system, as previously described in^{38 (link)}. For SkM-ELV miRNA profiling, 33 ng of total RNA were used for multiplex reverse transcription (RT). Pre-amplification was realized with 2.5 µl of cDNA using the TaqMan Megaplex Pre-Amp system (Applied Biosystems) according to manufacturer’s instructions. For Quad miRNA profiling, 900 ng were used for multiplex RT and the resulting cDNA was not pre-amplified. Then each cDNA reaction was mixed with TaqMan Universal PCR Master mix and loaded into the corresponding fill port. Individual singleplex PCR reactions were performed in 384-well reaction plates with Applied Biosystems 7900HT Fast Real-time PCR system. The level of miRNA was measured using Ct (threshold cycle) determined by RQ Manager. For each miRNA, the threshold cycle (Ct) was calculated by the ABI 7900 Sequence Detection System software. We used the mean expression level of all fully detected miRNAs for normalization. Comparison between groups were made by using the student t-test (p < 0.05) on normalized data, to select the differentially expressed miRNAs between ob/ob mice and wild type. Data are expressed in Ct values, which are inversely proportional to miRNA concentrations.