Whole transcriptome amplification kit

Real-time PCR was used to validate microarray gene expression data of six differentially modulated genes: Defensin Alpha 4 (DEFA4); TNF Alpha Induced Protein 6 (TNFAIP6/TSG-6); Fatty Acyl-CoA Reductase 2 (FAR2); Lymphotoxin beta (LTB); Granulocyte chemotactic protein 2 (GCP-2/CXCL6); Integrin Subunit Alpha X (ITGAX). Experiments were performed on the same cohort used in microarray experiments. Reverse transcription (RT) was performed using a Whole Transcriptome Amplification Kit (Sigma Aldrich, St Louis, MO, USA), optimized to amplify RNA from FFPE samples. Each RT reaction contained 250 ng of total RNA and the obtained cDNA was purified using a MinElute Purification Kit (Qiagen GmbH, Hilden, Germany)^{3 (link)}. Real-time PCRs were performed in triplicate using PrimeTime qPCR Primer assays (IDT) on 30 ng of diluted cDNA. Real-time PCR amplification reactions were performed in triplicate in 25 μl final volumes via SYBR Green (SensiMix SYBR Hi-ROX kit, Bioline). The β-actin gene amplification was used as a reference standard to normalize the target signal. Amplification specificity was controlled by a melting curve and the amount of mRNA target was evaluated using the comparative cycle threshold (ΔCt) method.

Before library construction, extracted nucleic acids were subjected to cDNA synthesis, and amplification was carried out using the Whole Transcriptome Amplification Kit (WTA2, Sigma-Aldrich, Darmstadt, Germany) as per manufacturer instructions. Amplified PCR products were then purified using the Wizard® SV Gel and PCR Clean-Up kit (Promega, Madison, WI, USA). The quantity and quality of the purified product were checked using a Qubit dsDNA high sensitivity assay kit with Qubit Fluorometer v3.0 (Thermo Fisher Scientific, Waltham, MA, USA).
The library construction was performed as a pool that contained four samples using the Illumina DNA Prep (Illumina, San Diego, CA, USA) as per kit instructions, starting with 250 ng of DNA as measured by Qubit (Invitrogen). The quality and quantity of the prepared library was assessed by the Australian Genome Research Facility (AGRF), Melbourne, Australia. The prepared library was normalised and pooled in equimolar quantities. The quality and quantity of the final pooled library were further assessed as described above before sequencing by the facility. According to the manufacturer’s instructions, cluster generation and sequencing of the pooled library was performed with read lengths of 150-bp paired-end on Illumina® NovaSeq chemistry.

Before library construction, extracted nucleic acids were subjected to cDNA synthesis, and amplification was carried out using the Whole Transcriptome Amplification Kit (WTA2, Sigma-Aldrich, Darmstadt, Germany) as per manufacturer’s instructions. Amplified PCR products were then purified using the Wizard SV Gel and PCR Clean-Up kit (Promega, Madison, WI, USA). The quantity and quality of the purified product were checked using a Qubit dsDNA high sensitivity assay kit with Qubit Fluorometer v3.0 (Thermo Fisher Scientific, Waltham, MA, USA).
The library construction was performed on the four pooled samples using the Illumina DNA Prep (Illumina, San Diego, CA, USA) as per kit instructions, starting with 250 ng of DNA as measured by Qubit (Invitrogen). The quality and quantity of the prepared libraries were assessed by the Australian Genome Research Facility, Melbourne, Australia. The prepared libraries were normalized and pooled in equimolar quantities. The quality and quantity of the final pooled libraries were further assessed as described above before sequencing by the facility. According to the manufacturer’s instructions, cluster generation and sequencing of the libraries were performed with read lengths of 150 bp paired-end on Illumina NovaSeq chemistry.