![Fig. 1. Quantitative RT-PCR for relative expression levels of p21 and p27 with endogenously expressed β-actin used as an internal control. (A) Schematic presentation of primer setting on p21, p27, and β-actin sequences. Thick lines indicate coding regions and thin lines represent truncated noncoding regions. Thick arrows indicate primers used in the PCR. The common primer (p27S) is derived from the identical region between p21 and p27 sequences, except for one mismatch in the p21 sequence. The composite primer (βASp27S) consisted of p27S in the 5′ part and β-actin sequence in the 3′ part. The common primer (p27S) is shared in amplification of the three PCR products, whereas p21AS, p27AS, and βS are specific to p21, p27, and β-actin sequences, respectively. (B) Autoradiographs showing kinetics of the PCR. An aliquot (5 μL, 0.1 μg RNA equivalent) of cDNA synthesized from Nalm-6 cells RNA was placed in 100 μL of PCR reaction volume with [-32P]dCTP (10 μCi) and was subjected to PCR as described in Materials and Methods. An aliquot (5 μL) of the reaction mixture was taken at the indicated cycle. Arrows indicate the PCR products corresponding to p21, p27, and β-actin. (C) [-32P]dCTP incorporation was plotted on a logarithmic scale over number of cycles of PCR. Each symbol denotes the following: (•) β-actin; (○) p27; (▵) p21. (D) Comparison between Northern analysis and RT-PCR. RNAs extracted from MEG-01s cells stimulated with TPA at 10 nmol/L were subjected to RT-PCR and to Northern analysis. The gel for PCR products was dried and exposed to an x-ray film for 17 hours at −80°C with an intensifying screen. The membrane for Northern analysis of β-actin and p21 was exposed to x-ray films at −80°C with intensifying screens for 1 day and 7 days, respectively. p27 mRNA was hardly detected by Northern analysis (data not shown). (E) The amount of p21 transcripts standardized to β-actin determined by Northern analysis and by RT-PCR. The signal ratio of p21 over β-actin at time 0 hour was defined as 1 U and the amounts of p21 transcripts standardized to β-actin are plotted on a logarithmic scale over time. Each symbol denotes the following: (○) Northern analysis; (•) RT-PCR.](https://ash.silverchair-cdn.com/ash/content_public/journal/blood/93/12/10.1182_blood.v93.12.4167/4/blod41205001w.jpeg?Expires=1722652523&Signature=lPVE8S-TWVspX2RgPVODCGvFXgbG6ZiXpX2pm-Mnre8iKOTPQqn5Bs0dtcwe0MeHUeHzzUY8Pdilzb94kvXOmFZdsxv-mE4FJRQ3cbUyyByfNLpAQNYu8wxxa1a73KwOyskRhwLpAHYio~MyelBugwRkx2KhLOGZi28avMH-H~rFXWwGCCHVAxwXyt03AdHrCZofLXa~bS8DVhT1yeCxIoD6DmXqd4LikewcKa2c37ILaNnjtBZuLWihtxa1wCqBu1p2zpvHFsuoD1tx8YNTiY6F50HoFv79FYiFLY6i-kJVdNVluI7Uuzlw49WfYeyq-6LZdMFWSBOGSq7pUZArVw__&Key-Pair-Id=APKAIE5G5CRDK6RD3PGA)
Quantitative RT-PCR for relative expression levels of p21 and p27 with endogenously expressed β-actin used as an internal control. (A) Schematic presentation of primer setting on p21, p27, and β-actin sequences. Thick lines indicate coding regions and thin lines represent truncated noncoding regions. Thick arrows indicate primers used in the PCR. The common primer (p27S) is derived from the identical region between p21 and p27 sequences, except for one mismatch in the p21 sequence. The composite primer (βASp27S) consisted of p27S in the 5′ part and β-actin sequence in the 3′ part. The common primer (p27S) is shared in amplification of the three PCR products, whereas p21AS, p27AS, and βS are specific to p21, p27, and β-actin sequences, respectively. (B) Autoradiographs showing kinetics of the PCR. An aliquot (5 μL, 0.1 μg RNA equivalent) of cDNA synthesized from Nalm-6 cells RNA was placed in 100 μL of PCR reaction volume with [-32P]dCTP (10 μCi) and was subjected to PCR as described in Materials and Methods. An aliquot (5 μL) of the reaction mixture was taken at the indicated cycle. Arrows indicate the PCR products corresponding to p21, p27, and β-actin. (C) [-32P]dCTP incorporation was plotted on a logarithmic scale over number of cycles of PCR. Each symbol denotes the following: (•) β-actin; (○) p27; (▵) p21. (D) Comparison between Northern analysis and RT-PCR. RNAs extracted from MEG-01s cells stimulated with TPA at 10 nmol/L were subjected to RT-PCR and to Northern analysis. The gel for PCR products was dried and exposed to an x-ray film for 17 hours at −80°C with an intensifying screen. The membrane for Northern analysis of β-actin and p21 was exposed to x-ray films at −80°C with intensifying screens for 1 day and 7 days, respectively. p27 mRNA was hardly detected by Northern analysis (data not shown). (E) The amount of p21 transcripts standardized to β-actin determined by Northern analysis and by RT-PCR. The signal ratio of p21 over β-actin at time 0 hour was defined as 1 U and the amounts of p21 transcripts standardized to β-actin are plotted on a logarithmic scale over time. Each symbol denotes the following: (○) Northern analysis; (•) RT-PCR.
