Journal: Stem cells (Dayton, Ohio)

Article Title: Adenovirus mediated alpha interferon (IFN-alpha) gene transfer into CD34+ cells and CML mononuclear cells.

doi: 10.1002/stem.150386

Figure Lengend Snippet: Figure 6. Southern blot analysis of PCR products obtained from total RNA isolated from CD34+ cells transfected with AdCMV-IFN- α gene. Lane 1, PCR products amplified from nontransfected (control) CD34+ cell RNA; lane 2, RNA obtained from CD34+ cells transfected with AdCMV-IFN-α after 24 h; lanes 3 and 4, RNA from CFU-GM clones after 12 and 14 days of culture, respectively; lanes 5 and 6, RNA from BFU-E clones after 12 and 14 days of culture. Total RNA was extracted from CD34+ cells, BFU-E or CFU-GM clones. IFN-α transcripts were amplified by PCR using 2 ml of 100 ml cDNA mixture of a total reaction volume of 25 ml. Aliquots of the reaction mixture (10 ml) were subjected to electrophoresis in a 0.8% agarose (Sigma; St. Louis, MO) gel. Nucleic acids were transferred to nitrocellulose by blotting and probed with IFN-α cDNA probes, (gift of Dr. Asano) using α-32d-CTP. Filters were exposed to x-ray for 48 h at –80°C.

Article Snippet: RNA is reverse-transcribed using the first strand cDNA synthesis kit from Clontech, Inc. (Palo Alto, CA).

Techniques: Southern Blot, Isolation, Transfection, Amplification, Control, Clone Assay, Electrophoresis

Journal: Stem cells (Dayton, Ohio)

Article Title: Adenovirus mediated alpha interferon (IFN-alpha) gene transfer into CD34+ cells and CML mononuclear cells.

doi: 10.1002/stem.150386

Figure Lengend Snippet: Figure 7. Southern blot analysis of PCR products obtained from total RNA isolated from CML hematopoietic stem cells (BMMNC) transfected with AdCMV-IFN-α gene. Lane 1, PCR products amplified from nontransfected (control) cell RNA; lane 2, RNA obtained from cells transfected with AdCMV-IFN-α after 24 h; lane 3, RNA from CFU-GM clones after 12 days; lane 4, CFU-GM clones after 14 days of culture; lane 5, no RNA was added; and lane 6, positive control. Total RNA was extracted from stem cells or CFU-GM clones by one single extraction. cDNA was synthesized from total RNA. IFN-α transcripts were amplified by PCR using 2 ml of 100 ml cDNA mixture of a total reaction vol- ume of 25 ml. Aliquots of the reaction mixture (10 ml) were sub- jected to electrophoresis in a 0.8% agarose (Sigma) gel. Nucleic acids were transferred to nitrocellulose by blotting and probed with IFN-α cDNA probes, using α-32P-CTP. Filters were exposed to x-ray for 48 h at –80°C.

Article Snippet: RNA is reverse-transcribed using the first strand cDNA synthesis kit from Clontech, Inc. (Palo Alto, CA).

Techniques: Southern Blot, Isolation, Transfection, Amplification, Control, Clone Assay, Positive Control, Extraction, Synthesized, Electrophoresis