Rna to cdna ecodry kit

To examine the presence of the mutation-specific BRAF-V600E mRNA in peripheral DC and distinct BM progenitors, single cell suspensions from BM and liver were stained for the appropriate surface markers, and individual cell subsets were purified using FACSAria (BD) directly into TRIzol reagent (Invitrogen). After RNA isolation and reverse transcription with RNA to cDNA EcoDry kit (Takara Bio Inc.), expression level of BRAF-V600E was assessed by probe-based mutation-specific qPCR using iTaq Universal Probes Supermix (Bio-Rad Laboratories) on a CFX384 Touch Real-Time PCR Detection system. In detail, reaction was performed with a final dilution of 500 nM of the reverse primer 5′-GTAGCTGGCCGGTCATCAGTTCG-3′, 500 nM of mutation-specific forward primer 5′-TAGGTGACTTTGGTCTAGCCACGGA-3′, which includes the specific nucleotide substitution of the T>A at the 3′ end and an additional mismatch nucleotide (A>G at the third position from the 3′ end), further enhancing the specific detection of mutated transcripts (strategy adapted from Schnittger et al., 2012 (link)), and 250 nM of the FAM-labeled hybridization probe 6-FAM-TCAGACGTGTATGCGTTTGGGATTG-MGBNFQ at an annealing temperature 68°C for 45 cycles. Data were normalized to total BRAF expression using the TaqMan Gene Expression Assay Mm01165837_m1 (Life Technologies) recognizing the common exon 13–14 of wild-type BRAF and BRAF-V600E.