Freshly amplified PCR products were cloned into plasmid pGEM-T vector (Promega) according to commercial instructions.
Five μg of total RNA were reverse transcribed using oligo dT 18 and SuperScript reverse transcriptase II (Invitrogen) according to commercial instructions.
poly(A -RNA wA -RNAified from total RNA wash the Oligotex mRNA mini Kit (Qiagen) according to commercial instructions.
Total RNA was extracted from different tissues by the TRIzol® reagent (Invitrogen) and DNase-I treated (Fermentas, St. Leon-Rot, Germany) according to commercial instructions.
Total RNA was extracted by the phenol/chloroform-LiCl method as previously described [ 55] and the samples were cleaned using the Plant RNA Extraction Kit (Qiagen, Hilden, Germany) following commercial instructions.
DCM preservative solution was diluted 10 times with sterilized PBS buffer, referring to careHPV™ which had approved commercial instructions for DCM samples.
Labeled cRNA was hybridized using the gene expression hybridization kit (Agilent) following commercial instructions onto 44 K custom-designed 60-mer N. attenuata Agilent microarray containing 43,533 sequences (see accession numbers)[ 28, 29].
Genomic DNA was removed by DNAse treatment following commercial instructions (Turbo DNase; Ambion, Europe), RNA was cleaned up with RNeasy MinElute columns (Qiagen, Hilden, Germany) and the RNA quality was checked with the RNA 6000 n kit (Agilent, Santa Clara, CA) using an Agilent 2100 Bioanalyzer.
The design team wanted Proto Man to sport uncovered hair, but they instead opted for an open-faced helmet illustration for both the television commercials and instruction manual.
Moreover, fresh tissue was used to extract protein, DNA and RNA according to commercial kits instructions.
