Gdna eliminator mini spin columns

Cells were cultured and treated in 8-well plates (SCC-1, SCC-9, CAL-27, SCC-6, SCC-47, SCC-104, SCC-90 and SCC-152) and washed with 3 × 2 ml PBS, with the exception of UM-SCC-6 which were washed only 1 × 2 ml, cells were scraped, and transferred in 300 μL RNA Later (cat: AM7021, ThermoFisher Scientific) to 1.7 mL centrifuge tubes. The tubes were vortexed, and 20 μL of sample was transferred to a new centrifuge tube. RNA purification was performed using the RNeasy Plus Mini Kit supplemented with gDNA Eliminator mini Spin Columns (Cat#: 74134 Qiagen) and QIAshredder (Cat#: 79656 Qiagen) per manufacturers guidelines. RNA was used to synthesize cDNA using the iScriptTM Reverse Transcription Supermix for RT-qPCR (Cat#: 1708841 BIO-RAD) as per the manufacturer’s guidelines. Following cDNA synthesis, samples were diluted with Nuclease-free H2O to 1 ng/μl and either stored at -20 °C or used directly for droplet digital PCR (ddPCR).

RNA was extracted with the RNeasy Plus Mini Kit (Qiagen, Hilden, Germany) from untreated embryos and those treated with PCP at 24 and 48 hours. Genomic DNA was removed from samples with gDNA eliminator mini spin columns (Qiagen, Hilden, Germany). iScript (Bio-Rad, Hercules, CA) was used to generate cDNA from each sample. Each cDNA sample was made by pooling ten random whole embryos per condition. SsoAdvanced Universal SYBR green mastermix (Bio-Rad, Hercules, CA) was used to amplify genes, with an Eppendorf realplex2 (Eppendorf, Hamburg, Germany) machine using primers for ef1α [21 (link)], runx1 [21 (link)], cmyb [21 (link)], and itga2b (commonly referred to as cd41) [21 (link)]. All PCR primers were validated previously and designed to span exons. Data were analyzed for relative expression change with ef1α as the reference gene. ΔΔCt was calculated by comparing the expression of the treated embryos to control embryos and to the reference gene, ef1α.

Total RNA from the cultured CD34+ cells in experimental and control groups were extracted using RNeasy Mini kit with gDNA Eliminator Mini Spin Columns (Qiagen, Venlo, the Netherlands). RNA samples were transcribed to cDNA using Tetro cDNA Synthesis kit (Bioline, Eveleigh, NSW, Australia). qRT-PCR was performed with the ABI PRISM 7500 Fast Real-Time PCR System (Applied Biosystems) using Fast SYBR green mastermix (Applied Biosystems) and relative quantification was performed using the comparative CT (2^−ΔΔCT) method with hGAPDH as the housekeeping gene. The KiCqStart™ primers used were all purchased from Sigma as follows: H_HIF1A_1, H_HK1_1, H_ATP6V1H_1, H_NDUFA_10_1, and H_GAPDH_1.