Taqman preamp kit

RNA enriched in miRNAs was extracted from PND3 ovaries using the ReliaPrep™ miRNA Cell and Tissue Miniprep System (Promega, Leiden, Netherlands) based on manufacturer’s instructions, and the tissue homogenization was facilitated by a pestle mixer. Moreover, the DNAse treatment step decreased the possibility of genomic DNA contamination. The measurement of RNA quantity and purity was performed on a NanoDrop spectrophotometer (Thermo Scientific, Merelbeke, Belgium).
The samples were reverse transcribed into cDNA using the TaqMan^® MicroRNA Reverse Transcription kit (Applied Biosystems™, Merelbeke, Belgium) and multiplexed Megaplex™ RT Primers (Applied Biosystems™, Merelbeke, Belgium) based on the manufacturer’s instructions. After reverse transcription, a pre-amplification step was followed using 2.5 µL from the cDNA product, TaqMan^® PreAmp kit (Applied Biosystems™) and Megaplex™ PreAmp Primers (Applied Biosystems™, Merelbeke, Belgium), as it was indicated in the manufacturer’s protocol. Then, the pre-amplified products were diluted in 100 µL of Tris-EDTA (TE) buffer (pH 8). The step of pre-amplification further increased the sensitivity of the method.

RNA was extracted from CD34⁺ cells using Direct-zol™ RNA Kits (Zymo, Irvine, CA, USA) and Trizol Reagent (Invitrogen Carlsbad, CA, USA), RNA quality was assessed by Multiskan™ GO Microplate spectrophotometer and the ratios of absorbance at 260 and 280 nm were assessed for all the samples. Only samples with RNA ratios within 1.9 were included in the present study. cDNA synthesis was made from purified total RNA by reverse transcription using High capacity cDNA Reverse Transcription RETROscript^® Kit (Thermo Fisher Scientific, Waltham, MA, USA) followed by a pre-amplification using Taqman PreAmp Kit (Applied Biosystems) according to the manufacturer’s instructions. qRT-PCR analysis was performed using TaqMan assays (Life Technologies, Grand Island, NY, USA) containing PCR primers and TaqMan probes according to the manufacturer’s instructions. Normalized expression values were determined using the ΔCt method. qRT-PCR data were normalized for the expression of GAPDH. qRT-PCR reactions were performed in triplicate in a 96-well format using an Applied Biosystems 7900HT fast real-time PCR instrument. Relative expression was calculated using the comparative threshold cycle method as previously described (23 (link), 24 (link)). For two-groupcomparisons, a Student’s t test was employed. Reported below are the main characteristics of the primers used:

Colonic biopsies from three different patients were washed with phosphate‐buffered saline and incubated in 5 ml of dissociation medium [30 mm EDTA in Dulbecco's modified Eagle's medium without Ca²⁺ and Mg²⁺, 0.5 mm dithiothreitol, and 2% RNAlater (Life Technologies)] for 10 min at room temperature. Tissue was shaken vigorously for 30 s to release individual crypts. Ten single crypts from each patient were hemisected with fine needles under a dissecting microscope, and individual hemicrypts were then aspirated. RNA was extracted, preamplified with the TaqMan PreAmp kit (Applied Biosystems), and reverse transcribed prior to quantitative polymerase chain reaction (PCR).