Syber select master mix

Total RNA from the bone marrow (BM), mononuclear cells, or in vitro culture cell samples, was isolated using TRI Reagent^® (Sigma) according to the manufacturer's protocol. cDNA was synthesized from 1 µg of total RNA with random hexamer primers using a High‐Capacity cDNA Reverse Transcription Kit (Applied Biosystems). Quantitative real‐time PCR (RT–qPCR) was performed in ABI Prism 7300 (Applied Biosystems) with Syber Select Master Mix (Applied Biosystems). The relative quantity of cDNA was estimated by the ∆∆CT method; data were normalized to GAPDH. Unless stated otherwise, SBSN ∆∆CT was normalized to the mean of the ‘Hematological malignancies’ group. Subsequently, fold change (FC) was estimated. Primers used were purchased from Sigma: hSBSN: 5′‐CAG GCT GGA AAG GAA GTG GAG A‐3′, 5′‐CTT GAT GGC TGG AAG ATC CGC T‐3′; GAPDH: 5′‐GTC GGA GTC AAC GGA TTT GG‐3′, 5′‐AAA AGC AGC CCT GGT GACC‐3′.

C33A cells were seeded in a 60 mm culture dish and transfected with 3 µg of each E6 plasmid. After 48 h post-transfection, cells were collected, and total RNA extraction was performed using the RNeasy mini kit (Qiagen, Hilden, Germany). The isolated RNA was treated with the DNAse Free DNA removal kit (Thermo Fisher Scientific, Waltham, MA, USA) and 400 µg of RNA was reverse-transcribed with random hexamers utilizing the GeneAmp RNA PCR Core Kit (Applied Biosystems, Foster City, CA, USA) For the Cyclin D1 amplification, forward 5′-ACAAACAGATCATCCGCAAACAC-3′ and reverse 5′-TGTTGGGGCTCCTCAGGTTC-3′ primers were used. For Sp5 amplification, forward 5’-TCGGACATAGGGACCCAGTT-3′ and reverse 5′-CTGACGGTGGGAACGGTTTA-3′. As a house keeping control, 18S mRNA was amplified with forward 5’-AACCCGTTGAACCCATT-3′ and reverse 5’-CCATCCAATCGGTAGTAGCG-3′ primers. SYBER select Master Mix (Applied Biosystems, Foster City, CA, USA) was utilized for qPCR reactions. For Axin2 amplification, Taqman probes were used (Applied Biosystems, Foster City, CA, USA): Axin2 FAM (Hs00610344_m1) and 18S VIC (Hs99999901_s1) probes, with Taqman Gene Expression Master Mix for qPCR analysis (Applied Biosystems, Foster City, CA, USA). The results are presented as relative quantification using the ^ΔΔCt method.