Quantitative Analysis of p16Ink4a Expression

Total cellular RNA was extracted from about 20,000 sorted HSCs using TRIzol reagent (Invitrogen, Carlsbad, CA) following the manufacturer’s instructions. RNA yield and quality were determined by measuring absorbencies at 260 and 280 nm, respectively. First-strand cDNA was synthesized from total RNA by RevertAid First Stand cDNA Synthesis Kit (Thermo scientific) according to the manufacturer’s protocol. Quantitative real-time PCR was performed using TaqMan PCR primers and MasterMix (Applied Biosystems). Taqman MGB probes for the p16Ink4a (catalog number: Mm00494449_m1), and- the housekeeping gene Gapdh (catalog number: Mm99999915_g1) were obtained from Applied Biosystems (Foster City, CA). All samples were analyzed in triplicate using an ABI Prism 7500 Sequence Detection System (Applied Biosystems). The threshold cycle (CT) values for each reaction were determined and averaged using TaqMan SDS analysis software (Applied Biosystems). The changes in gene expression were calculated by the comparative CT method (fold changes = 2^−△△CT) as described previously [41 (link)].