Abi sds 2

Total RNA from cell lines and PBMC from patients was extracted by using TRIzol reagent (Invitrogen). For miRNA amplification, RNA was reverse transcribed into single stranded complementary DNA (cDNA) using ±TaqMan MicroRNA Assays system (Applied Biosystems) and specific primer for hsa-miR-21 and RNU6B as control. mRNA was reverse transcribed into cDNA using M-MLV Reverse Transcriptase (Invitrogen) according to the manufacturer’s instructions.
Both mRNA and miRNA levels were detected by either TaqMan Assays (PTEN Hs02621230_s1, PDCD4 Hs00377253_m1, PIAS3 Hs00966035_m1) or TaqMan MicroRNA Assay (hsa-miR-21 000397) (Applied Biosystems). RNU6B (001093) and GAPDH (Hs03929097_g1) were used as internal controls for miRNA and mRNAs, respectively. Primary miR-21 was amplified with specific primers previously described (see ref.¹⁷ ) and quantified using SYBR GreenER qPCR SuperMix. QRT-PCR was performed in an ABI Prism 7900HT Sequence Detector System (Applied Biosystems) in triplicates for each cDNA. ABI SDS 2.4 Software (Life Technologies) was used to calculate relative expression, using comparative Ct method (ΔΔCt). RNA expression levels are defined as arbitrary units (AU) using Ramos cell line as a calibrator.

TaqMan gene primer sets were purchased from Life Technologies (LifeTechnologies, Carlsbad, CA, USA). Reverse transcription and PCR amplification for gene expression assays were performed with TaqMan Transcription and TaqMan Gene Expression Master Mix, respectively, following the manufacturer’s instructions (LifeTechnologies, Carlsbad, CA, USA). RT-qPCR data were normalized, using POL2RA as housekeeping gene.
miRNA RT-qPCR was performed using the miRCURY LNA Universal RT microRNA PCR system (Exiqon, Vedbaek, Denmark) according to the manufacturer’s instructions.
Total RNA (20 ng) was polyadenylated and reverse-transcribed at 42°C (60 min), in a reaction volume of 20 μl using a poly-T primer containing a 5′ universal tag. After heat-inactivation at 85°C the resulting cDNA was diluted 80-fold in nuclease free water, and a volume of 8 μl was amplified in 20 μl reaction volume as follows: 95°C for 10 min, followed by 40 cycles at 95°C for 10 s and 60°C for 60 s. Normalization was performed with SNORD48 [36 (link)].
Gene and miRNA expression levels were quantified, using a sequence detection system (ABI Prism 7900HT; LifeTechnologies, Carlsbad, CA), and the threshold cycle (Ct) for each sample was determined. ABI SDS 2.4 software (LifeTechnologies, Carlsbad, CA, USA) was used to recover the data, and the relative expression was calculated, using the comparative ΔCt method.