HalfHeartNormalized
RNA extraction:   Approximately 50-100mg of tissue was harvested and and stored in RNAlater (Ambion, AM7020). RNA was extracted using TRIzol Plus RNA Purification Kit (Invitrogen cat no: 12183-555) including an on column DNase digestion using PureLink DNase (Invitrogen 12185-010).  The RNA was quantitated using nanodrop then diluted 1:250 in TE and quality checked via use of an Agilent Pico Chip (5067-1513).    CDNA synthesis:   The cDNA samples were prepared using a modification of the protocol developed by Galina Aglyamova, Eli Meyer, Mikhail V. Matz, Jade Buchanan-Carter, Keithanne Mockaitis, and John K. Colbourne using the SMART PCR cDNA Synthesis Kit (Clontech, cat no: 634902).  For the first strand cDNA synthesis, triplicate 5uL reactions consisting of 1ug RNA, 1uL of 12uM Cap-Trsa-CV primer (AAG CAG TGG TAT CAA CGC AGA GTC GCA GTC GGT ACT TTT TTC TTT TTTV), 1uL of 12uM SMART II A Oligonucleotide ( AGG CAG TGG TAT CAA CGC AGA GTA CGC GGG ), and water, were incubated at 72C for 2 minutes, followed by a 2 minute incubation on ice. To each reaction, 5uL of 2uL 5X First-Strand Buffer, 1uL 20mM DTT, 1uL of 10uM each dNTP mix, and 1uL Invitrogen Superscript Reverse Transcriptase (Invitrogen, cat no: 18064-014 )were added and incubated at 42C for 1 hour.  The reaction was terminated by placing on ice. 16 100uL amplification reactions were carried out using 2uL of the above single stranded cDNA.  Each reaction consisted of 80uL water, 10uL 10X Advantage 2 PCR Buffer, 2uL 50XdNTP Mix, 4uL 12uM 5'PCR Primer IIA ( AAG CAG TGG TAT CAA CGC AGA GT ), 2uL 50X Advantage 2 Polymerase Mix (Clontech, cat no: 639201), and 2uL template.  The reactions were incubated at 95C for 1 minute followed by 15 cycles of 95C for 7 seconds, and 68C for 6 minutes (Hale, M.C., 2009).  The product was then purified using QIAGEN QIAquick PCR Purification Kit, and visualized using Agilent DNA 7500 Chip.  The cDNA is precipitated using 2 volumes ethanol and 1/10 volume 3M Sodium acetate pH 5.3 and resuspended in water to give a concentration of 2ug/12uL.  The normalization was conducted following the Evrogen Trimmer Kit Protocol (Evrogen, cat no: NK001).  A reaction mixture of 12uL cDNA, 4uL of 4X Hybridization Buffer, and 4uL water is aliquoted into 4uL individual reactions overlayed by mineral oil and incubated at 98C for 2 minutes and 68C for 5 hours.  5uL of heated DSN master buffer is added to each reaction and incubated at 68C for 10 minutes.  1uL of one of four different DSN treatments (DSN, 1/2 dilution DSN, 1/4 dilution DSN, 0 DSN) is added to each reaction and incubated at 68C for 25 minutes.  The reactions are stopped by the addition of 10uL of DSN stop solution and incubated for an additional 5 minutes at 68C then 20uL of water is added to each tube.  The optimally DSN treated cDNA sample (treatment with 1uL nondiluted DSN) is then amplified using thirty 30uL reactions with each reaction consisting of 23.48uL water, 3uL 10X Advantage 2 PCR Buffer, 0.75uL 50XdNTP Mix, 1.17uL 12uM 5'PCR Primer IIA ( AAG CAG TGG TAT CAA CGC AGA GT ), 0.6uL 50X Advantage 2 Polymerase Mix (Clontech, cat no: 639201), and 1uL template and incubated at 95C for 1 minute followed by 16 cycles of 95C for 7 seconds, and 68C for 6 minutes. The product was then purified using QIAGEN QIAquick PCR Purification Kit, and visualized using Agilent DNA 7500 Chip.  A sample of at least 5ug of normalized cDNA in 120-140uL TE was then handed to the Emory Genomics Core for cDNA Library Preparation and sequencing.