Rna extraction rneasy plus mini or micro kit

BM LSKs were purified from femurs and tibiae. Staining was performance as described above. Total RNA was prepared with RNA Extraction RNeasy Plus Mini- or Micro-kit (Qiagen). RNA was reverse-transcribed with High-Capacity cDNA Reverse Transcription reagents kit (Applied Biosystems) according to the manufacturer's protocol. Real-time quantitative PCR (SYBR-green, Applied Biosystems) assays were performed with an Applied Biosystems 7900HT Fast Real-Time PCR System sequencer detector. Expression was normalized to Hprt (F-5′-CCTAAGATGAGCGCAAGTTGAA-3′, R-5′-CCACAGGACTAGAACACCTGCTAA-3′) and 36b4 (F-5′-ACTGGTCTAGGACCCGAGAAG-3′, R-5′-TCCCACCTTGTCTCCAGTCT-3′) expression. The primer sequences for Glg-1 were: F-5′-CAAGATGACGGCCATCATTTT-3′, R-5′-TTCCCCAAGACGAATGCTGC-3′; for p18: F-5′-TTATGAAGCACACAGCCTGCAATGT-3′, R-5′-ACGGACAGCCAACCAACTAACGG-3′; for p21: F-5′-TGTCTTGCACTCTGGTGTCTGAGC-3′, R-5′-TCTTGCAGAAGACCAATCTGCG-3′; for p57: F-5′-GCGCAAACGTCTGAGATGAG-3′, R-5′-AGAGTTCTTCCATCGTCCGCT-3′; for Tgfb1: F-5′-CCGAAGCGGACTACTAT-3′; R-5′-GTAACGCCAGGAATTGT-3′.