Screening of colonies in the FB-subtracted library using southern hybridization. (A) The blot of FB274 to FB320 is shown as a representative to explain the screening strategy to isolate colonies of FB-subtracted library for further analysis. The nested PCR products of FB-subtracted library were ligated to TA vector and transformed into DH5a cells. Bacterial plasmids were extracted, restriction digested with EcoRI, and gel fractionated on 1.5% agarose gel. Plasmid DNA were then transferred to nylon membrane and hybridized with MPB-HSCs amplified cDNA that was subsequently digested with RsaI and used as a probe. Colonies with the least signal or no hybridization signal were selected for further analysis. (B) Verification of the screening strategy. One of the colonies, MTCO-1, that showed a high hybridization signal with the cDNA of MPB-HSPCs was selected to further analyze its expression in FB-HSPCs and MPB-HSPCs in a Q-PCR approach. (Bi) Using 400 pg of amplified cDNA of FB-HSPCs and MPB-HSPCs, it is shown that there is not dramatic difference between the expression profile of MTCO-1 in both populations (FB-HSPCs Ct = 21.0 and MPB-HSPCs Ct = 20.5). (Bii) However, Trim-28 as one of the colonies with low hybridization signal with cDNA of MPB-HSPCs turned out to be expressed more in FB-HSPCs (Ct = 26.5) than MPB-HSPCs (Ct = 29.0).