Computational protocol: Identification and dynamic changes of RNAs isolated from RALY-containing ribonucleoprotein complexes

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Protocol publication

[…] MCF7 cells were grown on 10 cm Petri dishes. Total RNA was extracted from four biological replicates using the Agilent Total RNA Isolation Mini kit (Agilent Technologies) according to the manufacturer's protocol. RNA was quantified using the NanoDrop spectrophotometer (Thermo Fisher) and the quality assessed by Agilent 2100 Bioanalyzer. Hybridization, blocking and washing were performed according to Agilent protocol ‘One-Color Microarray-Based Gene Expression Analysis (Quick Amp Labeling)’. Probes were hybridized on a chip of Agilent-039494 SurePrint G3 Human GE v2 8 × 60K Microarray. Hybridized microarray slides were then scanned with an Agilent DNA Microarray Scanner (G2505C) at 5-micron resolution with the manufacturer's software (Agilent ScanControl 8.1.3). The scanned TIFF images were analyzed numerically and the background was corrected using the Agilent Feature Extraction Software version 10.7.7.1 according to the Agilent standard protocol GE1_107_Sep09. The output of Feature Extraction was analysed with the R software environment for statistical computing (http://www.r-project.org/) and the Bioconductor library of biostatistical packages (http://www.bioconductor.org/). Low signal Agilent features, distinguished by a repeated ‘undetected’ flag across the majority of the arrays in every condition, were filtered out from the analysis, leaving features corresponding to 15 126 HGNC genes. Signal intensities across arrays were normalized with the quantile method. Differentially expressed genes (DEGs) were identified using linear models and moderated t-test as implemented in the Bioconductor Limma package, using a double threshold on the log2 fold change (absolute value > 0.75) and the correspondent statistical significance (P-value < 0.05). [...] Total RNA was purified using TRIzol (Thermo Fisher) according to the manufacturer's protocol. The total RNA concentration and purity of samples were assessed using Nanodrop spectrophotometer (Thermo Fisher). Next, 1 μg of RNA was subjected to DNase (Thermo Fisher) treatment and retro-transcribed using the RevertAid First Strand cDNA Synthesis Kit (Thermo Fisher) in 20 μl-reaction, according to manufacturer's instructions. Then, the quantification of transcripts in the samples was performed by real time qRT-PCR analysis using TaqMan probes and it was carry out in CFX96 Touch™ Real-Time PCR Detection System (Bio-Rad). Briefly, the qPCR reaction (10 μl) contained: 1× KAPA PROBE FAST, 1 μM primers plus TaqMan probe and 2 μl of cDNA template obtained in the previous step, diluted 1:4. The qPCR assay was performed with the following amplification program: 95°C for 3 min, followed by 40 cycles of 95°C for 10 s and 60°C for 30 s, followed by a hold at 4°C. Primers and TaqMan probes for ACTB, ANXA1, CST6, CXCR4, GAPDH, H1FX, RALY and TMCO1 transcripts were purchased by IDT (TEMA Ricerca) (see ). The results were analyzed with the Bio-Rad CFX Manager version 2.1. The relative expression was calculated according to the 2−ΔΔCt method. Each reported experiments was performed, at least, in biological triplicates and technical duplicates. […]

Pipeline specifications

Software tools CFX Manager, ddCt
Application qPCR
Organisms Homo sapiens