Deoxyribonucleic acid dna free dna removal kit

Ribonucleic acid (RNA) was isolated from ALDEFLUOR positive and negative sorted cells that were cultured at high and low densities. RNA was isolated using the TRIzol reagent (Thermo Fisher Scientific) and the protocol provided by the manufacturer. Briefly, cells were pelleted and lysed in TRIzol reagent. After a brief incubation at RT, chloroform was added and samples were incubated for 2–3 minutes at RT. Samples were centrifuged and the aqueous phase was removed. RNA was precipitated with isopropyl alcohol, washed with ethanol and resuspended in sterile water. RNA was Deoxyribonuclease (DNAse) treated with the Deoxyribonucleic acid (DNA)-free DNA Removal Kit (Ambion) per the manufacturer’s protocol. Concentration of the RNA was determined using the Tecan Group Ltd. (TECAN) Infinite 200 PRO microplate reader. Equal amounts of RNA were used for the reverse transcriptase reaction. Using the SuperScript III First-Strand Synthesis System (Life Technologies) and the provided protocol, complementary DNA (Cdna) was generated. Polymerase Chain Reaction (PCR) was performed using 50 ng cDNA and the GoTaq Green PCR Mastermix (Promega). Primers and reaction conditions for the 19 ALDH isoforms were previously published.^{17 (link)} PCR products were analyzed on a 1.5% agarose gel and imaged on the Syngene imaging system.