Purelink rna mini isolation kit

Total RNA was extracted using PureLink RNA mini isolation kit (by Ambion Life Technologies, Carlsbad, CA, Cat No: 12183018 A) as recommended by the manufacturer. One hundred and fifty nanograms of total RNA were labeled and then hybridized to the Agilent Human SurePrint G3 Human GE 8 × 60 k microarray chip (Agilent Technologies, Santa Clara, CA). All microarray experiments were conducted at the Microarray Core Facility (Stem Cell Unit, King Saud University College of Medicine). Normalization and data analyses were conducted using GeneSpring GX software (Agilent Technologies). Pathway analysis was conducted using the Single Experiment Pathway analysis feature in GeneSpring 12.0 (Agilent Technologies) as previously described^{21 (link)}. A two fold cutoff with P < 0.02 was used.

Total RNA was isolated using Ambion’s PureLink RNA Mini Isolation kit according to the manufacturer’s instructions. RNA samples designated for RNA Sequencing were eluted in 30 µL filtered, autoclaved Mill-Q water. Subsequently, 2 ng of RNA was reverse transcribed into cDNA using BioRad’s iScript kit or BioRad’s iScript SuperMix. All cDNA was normalized to 350 or 500 ng (for BDNF mRNA assays) following conversion. The relative mRNA expression levels were determined using real-time quantitative PCR by General Taqman PCR master mix and TaqMan specific probes. Relative mRNA expression levels were determined by the ddCt method, with each normalization indicated where appropriate.

The qPCR was used to determine the level of Galnts gene expression in livers from wild-type and Galnt2^−/− mice. DNase-free RNA was isolated using the PureLink® RNA Mini Isolation Kit (Ambion). cDNA synthesis was performed using the iScript cDNA Synthesis Kit (Bio-Rad). PCR primers used were published previously [23 (link)] using Beacon Designer software (BioRad). The qPCR was performed on a CFX96 real time PCR thermocycler (Bio-Rad) using the SYBR-Green PCR Master Mix (Bio-Rad). The qPCR was performed in triplicate, and four independent experiments were performed. Gene expression levels were normalized to 29S rRNA and displayed as relative expression levels.

Total RNA was extracted from differentiating cells either treated or not treated (control samples) with single dose of TGF-β1 at D5 of induction using a PureLink RNA mini isolation kit (Cat No: 12183018 A, Ambion by Life Technologies, Austin, TX) as recommended by the manufacturer. Total RNA was quantified using a Nanodrop spectrophotometer (Nanodrop 2000, Thermo Scientific, Waltham, MA). cDNA was synthesised from 1 μg of the RNA samples using a High Capacity cDNA Reverse Transcription kit (Applied Biosystems, Foster City, CA) using a Labnet, Multigene thermocycler (Edison, NJ) according to the manufacturer’s instructions. Relative levels of mRNA were determined from cDNA using real time PCR (Applied Biosystem-Real Time PCR Detection System) with a Power SYBR Green PCR kit (Applied Biosystems, UK), or with the TaqMan Universal master Mix II, no UNG (Applied Biosystems, Foster City, CA) according to the manufacturer’s instructions. Following normalisation to the reference gene GAPDH, quantification of gene expression was carried out using a comparative Ct method where ΔCT is the difference between the CT values of the target and reference gene. Primers (Supplementary Tables S8 and S9) were either obtained directly from Applied Biosystems (Foster City, CA) as TaqMan primers or were synthesised by Life Technologies based on previously published primer sequences.

Total RNA was extracted using PureLink RNA Mini Isolation Kit (Ambion by Life Technologies, USA, Cat. No.: 12183018A) as recommended by the manufacturer. One hundred and fifty nanograms of total RNA were labeled and then hybridized to the Agilent Human SurePrint G3 Human GE 8 × 60 k v16 microarray chip (Agilent Technologies, Santa Clara, CA, USA). All microarray experiments were conducted at the Microarray Core Facility (Stem Cell Unit, King Saud University College of Medicine). Normalization and data analyses were conducted using the GeneSpring GX software (Agilent Technologies). Pathway analysis was conducted using the Single Experiment Pathway analysis feature in GeneSpring 12.0 (Agilent Technologies) as described in (ref. 5). Twofold cutoff with P<0.02 was used.