American Society of Hematology

Figure 3.

$Figure 3. Efficient SSH between FB-HSPCs versus MPB-HSPCs. The amplified cDNA of FB-HSPCs was subtracted from excess amount of MPB-HSPCs in a 2-step hybridization at 68°C to remove equally expressed genes. The differentially regulated genes in subtracted and unsubtracted libraries were amplified in suppression and nested PCRs. (A) In order to evaluate the quality of subtracted library, 50 ng of FB-subtracted and FB-unsubtracted DNA was used as a template to assess β-actin amplification in a semiquantitative PCR. An aliquot (7 mL) of amplified product was taken at 15 cycles and after every 5 cycles to 40 cycles followed by 2% agarose gel fractionation to estimate the abundance of β-actin at different cycles. (B) Q-PCR analysis of GAPDH, as one of the other housekeeping genes, verified the quality of FB-subtracted by using 10 ng of DNA from FB-subtracted (Ct = 36; ▦) and FB-unsubtracted (Ct = 23; ♦) libraries. (C) Amplification of Tal-1 as one of the key transcription factors involved in the occurrence of hematopoiesis. Semiquantitative PCR, the same methodology explained in Figure 2A, using 20 ng of amplified cDNA from FB-HSPCs and MPB-HSPCs, indicated equal expression of Tal-1 in both populations (Figure 2C top). In the FB-subtracted library, however, using 50 ng of DNA from FB-subtracted and FB-unsubtracted libraries, Tal-1 amplicon is detectable after 40 cycles in the FB-subtracted library as opposed to its appearance only after 15 cycles in the FB-unsubtracted library. (D) Amplification of E2F1 as one of the critical transcription factors involved in the cycling cells indicated a moderately higher expression in FB-HSPCs than MPB-HSPCs (Figure 2D top). In a semiquantitative approach, using 50 ng DNA as template, E2F1 is detectable in FB-HSPCs after 15 cycles but is present in MPB-HSPCs after 20 cycles. However, in an efficient SSH (Figure 2D bottom), using 100 ng DNA of subtracted and unsubtracted libraries, E2F1 is detected after 35 cycles in FB-subtracted but is observed at 40 cycles in the FB-unsubtracted library, indicating efficient SSH to enrich differentially regulated genes in FB-HSPCs.$

Efficient SSH between FB-HSPCs versus MPB-HSPCs. The amplified cDNA of FB-HSPCs was subtracted from excess amount of MPB-HSPCs in a 2-step hybridization at 68°C to remove equally expressed genes. The differentially regulated genes in subtracted and unsubtracted libraries were amplified in suppression and nested PCRs. (A) In order to evaluate the quality of subtracted library, 50 ng of FB-subtracted and FB-unsubtracted DNA was used as a template to assess β-actin amplification in a semiquantitative PCR. An aliquot (7 mL) of amplified product was taken at 15 cycles and after every 5 cycles to 40 cycles followed by 2% agarose gel fractionation to estimate the abundance of β-actin at different cycles. (B) Q-PCR analysis of GAPDH, as one of the other housekeeping genes, verified the quality of FB-subtracted by using 10 ng of DNA from FB-subtracted (Ct = 36; ▦) and FB-unsubtracted (Ct = 23; ♦) libraries. (C) Amplification of Tal-1 as one of the key transcription factors involved in the occurrence of hematopoiesis. Semiquantitative PCR, the same methodology explained in Figure 2A, using 20 ng of amplified cDNA from FB-HSPCs and MPB-HSPCs, indicated equal expression of Tal-1 in both populations (Figure 2C top). In the FB-subtracted library, however, using 50 ng of DNA from FB-subtracted and FB-unsubtracted libraries, Tal-1 amplicon is detectable after 40 cycles in the FB-subtracted library as opposed to its appearance only after 15 cycles in the FB-unsubtracted library. (D) Amplification of E2F1 as one of the critical transcription factors involved in the cycling cells indicated a moderately higher expression in FB-HSPCs than MPB-HSPCs (Figure 2D top). In a semiquantitative approach, using 50 ng DNA as template, E2F1 is detectable in FB-HSPCs after 15 cycles but is present in MPB-HSPCs after 20 cycles. However, in an efficient SSH (Figure 2D bottom), using 100 ng DNA of subtracted and unsubtracted libraries, E2F1 is detected after 35 cycles in FB-subtracted but is observed at 40 cycles in the FB-unsubtracted library, indicating efficient SSH to enrich differentially regulated genes in FB-HSPCs.

This Feature Is Available To Subscribers Only