(SuperScript III 1 st strand cDNA synthesis kit, Invitrogen) ..
The TRIzol reagent and SuperScriptChoice System cDNA synthesis kit were purchased from Invitrogen(Carlsbad, CA, USA). The BioArray high-yield RNA transcriptlabeling kit was obtained from Enzo Biochem (New York, NY, USA).The RNeasy kit was supplied by Qiagen (Valencia, CA, USA). TheCanine 2.0 and Mouse 430A 2.0 GeneChips were provided by Affymetrix(Santa Clara, CA, USA). GeneChip hybridization and scanning wereperformed at the Seoulin Molecular Biology Technique Center (Seoul,Korea).
cDNA RNase H ds cDNA for use ..
RNA isolation was performed using RNeasy (Qiagen) and RNA quality was assessed using a Bioanalyzer (Agilent). Purified total RNA was briefly treated (15 min at room temperature) with 0.5 units of DNase I (AMP grade; Invitrogen) per µg of RNA to remove residual DNA, followed by ethanol precipitation and purification of RNA. Double stranded cDNA was synthesized using SuperScript Double-Stranded cDNA Synthesis Kit (Invitrogen) according to NimbleGen protocols, followed by phenol:chloroform:isoamylalcohol (25:24:1), chloroform:isoamylalcohol (24:1) backextraction and ds cDNA was recovered by ethanol precipitation. Typically, 4-10 µg of input total RNA yielded 3-9 µg of ds cDNA. First strand cDNA synthesis and quantitative RT-PCR were performed as previously described (Campsteijn , 2012), and primer pair specificities were evaluated by melting curve assessment and amplicon sequencing. All transcript levels were normalized to RPL23 and EF-1-beta.
1st strand cDNA synthesis was performed using SuperScript III (Invitrogen) and the library was amplified using the Kapa HiFi Library Amp real time Kit (Peqlab) with Illumina TruSeq compatible primers.