Gdna eliminator

There were a total of 52 samples (31 AMI and 21 controls) available for transcriptome analysis. Blood samples were collected in EDTA tubes within 48-hours of AMI or following recruitment into the study. Nucleated cells were fractionated from 5 mL of heparinized blood. Total RNA was extracted from cell populations using a combination of gDNA Eliminator and RNeasy columns (Qiagen, Valencia, CA) and was assessed for quality and quantification using Agilent bioanalzyer and OD260/OD280 ratio. Biotinylated cRNA was prepared according to the standard Affymetrix protocol from 100ng total RNA. Following fragmentation, 120ug of cRNA was hybridized for 16 hours at 45°C on Affymetrix GeneChip Human Genome U133 Plus 2.0, which includes 54,675 probsets (http://www.affymetrix.com/estore/), with one sample per array. GeneChips were washed and stained in the Affymetrix Fluidics Station 450 and subsequently scanned using the GeneChip Scanner 3000 7G.

Gene expression was probed by RT-PCR. Total RNA was extracted using gDNA Eliminator and RNeasy columns (Qiagen, Valencia, CA). cDNA was prepared using Superscript III First Strand (Invitrogen).^{28 (link)} Mouse Gapdh was used as normalizing control. Primers used included predesigned Sox2 (Mm00488369_s1), Pou5f1 (Mm00658129_gH), Klf4 (Mm00516105_g1), Myc (Mm00487804_m1), Fgf4 (Mm00438917_m1), Gsc (Mm00650681_g1), Lhx1 (Mm00521776_m1), Flk-1 (Mm00440099_m1), Cxcr4 (Mm01292123_m1), αMHC (Mm01313844_mH), βMHC (Mm01319006_g1), Actin (Mm01333821_m1), cTnI (Mm00437164_m1) and Gapdh (4352932E) from Applied Biosystems, as well as AFP (Mm.PT.49.10942304), Hnfa (Mm.PT.49.5290569), Nes (Mm.PT.49.12610703), Pax6 (Mm.PT.49.14285402), Sox1 (Mm.PT.49.17703872.g) and Gapdh (Mm.PT.49.39a.1) from iDT. Primers used for semi-quantitative detection of endogenous and exogenous levels of the reprogramming factors are presented in Supplemental Table 1.