Abi 7900 machine real time pcr system

Isolation of RNA was performed using the miRNeasy Mini Kit (Qiagen). An on-column DNase (Qiagen) digestion was performed according to manufacturer’s instructions. RNA was quantified by a NanoDrop spectrophotometer (Thermo Scientific). Reverse transcription was carried out using the iScript cDNA Synthesis Kit (Bio-Rad). Realtime PCR was performed using the ABI 7900 Machine Real-Time PCR System and SYBR Green PCR Master Mix (Applied Biosystems). The following primers were used for quantitative RT-PCR:
Ccna2 forward 5’-TGGATGGCAGTTTTGAATCACC-3’
Ccna2 reverse 5’-CCCTAAGGTACGTGTGAATGTC-3’
Ubc forward 5’- GAGTTCCGTCTGCTGTGTGA-3’
Ubc reverse 5’- CCTCCAGGGTGATGGTCTTA-3’

Erythroid cells were collected at specified time points using QIAzol (Qiagen) and total cellular RNA was isolated using the miRNeasy minikit (Qiagen) according to the manufacturer’s protocol. Reverse transcription was carried out using SuperScript II Reverse Transcriptase (Invitrogen). Relative transcript levels of different genes were quantified by real-time PCR, using SYBR Green (Applied Biosystems) and the ABI 7900 Machine Real-Time PCR system. Results were normalized to 18S (criteria for differences in 18S Ct values for control and experimental sample set at <1.5). Primer sequences are found in Supplemental Table 1.
Microarray experiments were performed and processed using the Affymetrix Mouse Genome 430 2.0 array at the Whitehead Institute Genome Core.