Taqman array human microrna a b cards

For each chemical treatment and control, 3 biological replicates were performed for miRNA quantification using TaqMan Array Human MicroRNA A + B Cards Set v3.0, which contains a primer set for 754 human miRNA sequences and was used according to the manufacturer’s instructions (Thermo Fisher Scientific, Waltham, Massachusetts). Synthesis of cDNA was carried out from 6 µl of RNA solution (3 µl for each primer pool) with Megaplex RT primers (pool A v.2.1 and pool B v.3.0) and TaqManTM MicroRNA Reverse Transcription Kit (Thermo Fisher Scientific). cDNA was amplified by use of Megaplex PreAmp primers pool A v.2.1 and pool B v.3.0 and TaqManTM PreAmp Master Mix. The amplified cDNA was mixed with TaqMan Universal PCR Master Mix (No AmpErase UNG). The obtained sample was loaded to TaqMan Array Human MicroRNA A + B Cards subjected to quantitative PCR analysis by use of 7900HT Fast Real-Time PCR system (Thermo Fisher Scientific). Thermal cycling condition was 2 min at 50.0°C, 10 min at 94.5°C, 40 cycles at 97.0°C for 30 s, and at 59.7°C for 1 min.

Total RNAs were extracted from three SIRT1‐silenced (si‐SIRT1) skin primary fibroblasts and three control fibroblasts. The potential miRNAs were detected by using TaqMan Array Human MicroRNA A + B Cards (Thermo Scientific). This microarray could quantify 754 human miRNAs (Table S1). Three endogenous controls (U6, RNA44 and RNA48) and one non‐human negative control were also included in this microarray. The experiment was performed following the manufacturer's instructions. In brief, each fill reservoir of the card was loaded with 100 μL of prepared PCR reaction mix, including 450μl PCR Master Mix, 6μl cDNA and 444μl Nuclease‐fee Water. Then, set up and run the real‐time PCR instrument (7900HT Fast Real‐Time PCR System). Experimental conditions were as follows: 94.5°C, 10 minutes, 1 cycle for enzyme activation; 97.0°C, 30 seconds, 40 cycles for denature; 59.7°C, 60 seconds, 40 cycles for anneal/extend.

In the pilot and multicenter studies, we obtained tumor tissue samples from 40-80 μm cuts of the paraffin block that contained the diagnostic specimen and placed them in 1.5 mL Eppendorf tubes, in duplicate. Total RNA was obtained from tumor slides using the Recover All total nucleic acid isolation kit (Applied Biosystems, Foster City, CA, USA). Four EBV⁺DLBCLe and four EBV–DLBCL cases were analyzed in this part of the pilot study. We obtained cDNAs using 10ng of total RNA without preamplification and Megaplex Pools for miRNA expression (Applied Biosystems). The cDNA was inserted into two platforms (TaqMan Array Human microRNA A+B Cards) containing 384 human miRNAs each (TaqMan low-density arrays) on 7900 Real Time PCR System (Applied Biosystems). We considered miRNAs to be differentially expressed when the foldchange was above or below 1.5. The normalization method was performed using the endogenous RNU48 identified as the most stable among samples by software Normfinder (http://www.moma.dk/normfinder-software) and RNU6 recommended by the manufacturer, in a comparative way.