Primer sets for the following genes were purchased from SuperArray Bioscience Corporation (Frederick, MD, http://www.superarray.com): Rex3 (Mm.14768; PPM25806A; position 595–613; band size 162 bp); Vdac1 (Mm.3555; PPM04115A; position 582–604; band size 118 bp); Ets1 (Mm.292415; PPM03600A; position 509–529; band size 151 bp); Pcbp4 (Mm.286394; PPM37494A, position 623–642; band size 90 bp); Myo10 (Mm.60590; PPM29651A, position 5,298–5,316; band size 179 bp); H1fx (Mm.33796; PPM28101A, position 128–147; band size 129 bp); Thop1 (Mm.26995; PPM26958, position 2,128–2,147; band size 120 bp); Msx2 (Mm.1763; PPM03170A, position 903–925; band size 149 bp); Cryab (Mm.178, PPM03570A, position 503–523, band size 192 bp); Vars2 (Mm.28420, PPM03327A, position 3,665–3,683; band size 191 bp); Peg10 (Mm.320575, PPM24997A, position 608–628, band size 203 bp); Calr (Mm.1971, PPM05020, position 1,527–1,547, band size 169 bp); Crmp1 (Mm.290995, PPM37854A, position 1,804–1,822, band size 144 bp); Ube4b (Mm.288924, PPM37643A, position 4,160–4,179, band size 81 bp); Pygo2 (Mm.22521, PPM26308A, position 1,428–1,448, band size 160 bp); 5730449L18Rik (Mm.21065, PPM26127A, position 445–467, band size 136 bp); AU041707 (Mm.200898, PPM32982A, position 1,890–1,911, band size 83 bp); Nes (nestin; Mm.331129; PPM04735; position 915–936; band size 100 bp); Tebp (telomerase binding protein, p23; Mm. 305,816; position 881–903; band size 145 bp). Primers sets for the following genes (according to Rendl et al. ; their supplemental Table S14) were purchased from Operon Biotechnologies, Inc. (Huntsville, AL, http://www.operon.com): Akp2: 5′-TGCGC-TCCTTAGGGCTGCCG-3′, 5′-GGTGTACCCTGAGATTCGTCC-3′, band size 160 bp; Hoxa9: forward: 5′-GTTCTCCGGGATGCATAGATTCA-3′, reverse: 5′-GAACCCCAAA-TTCA-CCAAGGATAC-3′, band size 346 bp; Zic1: forward: 5′-GC-GGCCGAAAGCCAACT-3′, reverse: 5′-TGCCAAAAGCAA-TGGACAGC-3′, band size 315 bp. Cd34 primers were used according to Drew et al. : forward 5′-CTCTAGATCACAAGTTCTGTGTCAGC-3′, reverse 5′-TAGCACAGAACTTCCCAGCAAAC-3′; hypoxanthine guanine phosphoribosyl transferase (Hprt) primers with the following sequences were used for normalization: 5′-CCTGCTGGATTACATTAAAGCACTG-3′ and 5′-CCTGAAGTACTCATTATAGTCAAGG-3′ (band size 350 bp).
For RT, total RNA was extracted with TRIzol reagent and treated with DNase (Invitrogen) to remove traces of genomic DNA. First-strand cDNA was synthesized using the SuperScript III First-strand synthesis system for RT-PCR (Invitrogen) and primed with oligo(dT) according to manufacturer's instructions. For quantitative PCR, each 25-μl of PCR mix consisted of 12.5 μl of 2× RT2 Real-Time SYBR Green/Fluorescein PCR Master Mix, 1.0 μl of First Strand cDNA template, and 1.0 μl of RT2 PCR Primer Set and was brought to a final volume of 25 μl using ddH2O. Thermocycling was performed as follows: 95°C, 15 minutes and 38 cycles of 95°C, 30 seconds; 55°C, 30 seconds; and 72°C, 30 seconds. The amplification rate of each target was evaluated from the cycle threshold (Ct) numbers obtained for serial cDNA dilution. For a given target, the Ct difference was calculated and then expressed as relative mRNA level. For each PCR product, a single narrow peak was obtained by melting curve analysis at the specific melting temperature and only a single band of the predicted size was observed by agarose gel electrophoresis. For conventional PCR, 12.5 μl of 2× ReactionReady HotStart “Sweet” PCR Master Mix, 1.0 μl of First Strand cDNA template, and 1.0 μl of RT2 PCR Primer Set were combined and brought to a final volume of 25 μl using ddH2O. Thermocycling was performed as follows: 95°C, 15 minutes and 30 cycles of (95°C, 30 seconds; 55°C, 30 seconds; and 72°C, 30 seconds). Ten-microliter PCR product was separated on a 1.8% agarose gel containing ethidium bromide.