7900ht fast instrument

The cDNA reaction mixture was diluted in water and cDNA equivalent of 25 ng RNA used for each sample. Quantitative real‐time PCR was performed with reagents and instruments from Applied Biosystems in the 96‐well format using a 7900HT Fast instrument and the SDS 2.3 software (Applied Biosystems). Predeveloped primers and probe sets (TaqMan assays; Applied Biosystems) were used to analyze mRNA levels of 13 genes for comparison between RT‐PCR, microarray and RNA sequencing. These 13 genes included secreted frizzled‐related protein 4 (SFRP4, Hs00180066_m1), leptin (LEP, Hs00174877_m1), OPG (TNFRSF11, Hs00900358_m1), interleukin‐6 (IL6, Hs00985639_m1), adiponectin (ADIPOQ, Hs00605917_m1), Apelin (APLN, Hs00936329_m1), PR domain containing 16 (PRDM16, Hs00922674_m1), T‐box transcription factor (TBX1, Hs00271949_m1), trans‐membrane protein 26 (TMEM26, Hs00415619_m1), Tumor Necrosis Factor Receptor Superfamily, Member 9 (TNFRSF9, Hs00155512_m1), Fibronectin type III domain‐containing protein 5 (FNDC5, Hs00401006_m1), Peroxisome Proliferator‐Activated Receptor Gamma, Coactivator 1 Alpha (PPARGC1A, Hs01016719_m1), and Uncoupling Protein 1 (Mitochondrial, Proton Carrier) (UCP1, Hs00222453_m1). Relative target mRNA expression levels were calculated as 2^−ΔCt, and normalized to beta‐2 microglobulin (B2M, Hs00984230_m1).

Using High‐Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Foster City, CA), 1000 ng of total RNA from human and mouse tissue samples or cells was converted into cDNA. The cDNA reaction mixture was diluted in water and an equivalent of 25 and 50 ng was analyzed in each sample in the mouse and human tissues, respectively. Quantitative real‐time PCR was performed in the 96‐well format using a 7900HT Fast instrument and the SDS 2.3 software (Applied Biosystems) (Haugen et al. 2010 (link)). Predesigned commercial primers and probe sets (TaqMan assays, Applied Biosystems) were used to analyze mRNA levels of ANGPTL4 (Hs00401006_m1), Angptl4 (Mm00480431_m1), beta‐2 microglobulin (B2M, Hs00984230_m1), large ribosomal protein P0 (RPLP0, Hs99999902_m1), and TATA box binding protein (Tbp, Mm00446973_m1). Relative target mRNA expression levels were calculated as 2^−ΔCt by normalizing to B2M and RPLP0 in humans and TBP in mice.

Total RNA reverse transcription and TaqMan Real Time RT-qPCR were performed as previously described [18 (link)] Relative expression levels were achieved by using the comparative cycle threshold (C_t) method of relative quantitation using GAPDH as the housekeeping gene. To normalize data, ΔΔC_t was calculated for each sample using the mean of its ΔC_t values subtracted from the mean ΔC_t value measured in the NegCTR mimic/NegCTR siRNA sample, set as a calibrator; relative quantitation (RQ) value was expressed as 2^−ΔΔCt. LEF1/NR4A2 relative expression kinetics during erythroid, megakaryocyte, granulocyte or monocyte/macrophage differentiation were similarly calculated by setting CD34+ cells as calibrator.
The miR-34a-5p expression levels were detected by RT-qPCR by means of TaqMan MicroRNA assays specific for miR-34a-5p and U6 snRNA, respectively (Thermo Fisher Scientific, Waltham, MA, USA). Five ng of total RNA were reverse-transcribed by using miRNA-specific looped-primers. Real Time RT-qPCR was performed by means of a 7900HT Fast Instrument (Applied Biosystems, Foster City, CA, USA). U6 snRNA was set as housekeeping control to assess miR-34a-5p Relative Quantity (RQ).