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. 1997 Mar 3;185(5):805–816. doi: 10.1084/jem.185.5.805

Figure 6.

Figure 6

MDM express CC–CKR-5 mRNA. Total RNA was extracted from untreated MDM. RNA samples were treated with DNase I to remove traces of contaminating DNA and reverse transcribed using random hexameric primers. The cDNA products were mixed to scalar amounts of a synthetic competitor DNA fragment containing primer recognition sites for both β-actin and CC–CKR-5 amplification, and amplified with the respective primer pairs. (A) Schematic representation of the competitor DNA fragment used for the quantification of CC–CKR-5 and β-actin cDNA. The fragment contains a core sequence derived from the human β-actin cDNA, carrying a 20-bp insertion in the middle (closed box). Amplification with the β-actin-specific primer set BA1–BA4 detects a 226-bp product on human cDNA, and a 246-bp product from the competitor DNA. To this core sequence, the primer recognition sites for human CC–CKR-5 amplification were added at the two ends (indicated by gray boxes) by reamplification with composite primers corresponding to the CKR-9+BA1 sequence at one end and CKR-10+BA4 at the other end. Amplification with CKR-9 and CKR-10 generates a 288-bp fragment from the competitor template and a 368-bp fragment from the CC–CKR-5 cDNA. (B) Competitive PCR for the quantification of CC– CKR-5 and β-actin mRNAs. cDNA samples from untreated MDM were mixed with tenfold dilution of the competitor DNA fragment as indicated, and amplified with primer sets CKR-9/CKR-10 and BA1/BA4 for CC–CKR-5 and β-actin mRNA quantification. Amplification products were resolved by polyacrylamide gel electrophoresis, stained with ethidium bromide, and quantified by densitometric scanning. According to the principles of competitive PCR, quantification of the target molecules in the samples was obtained by estimation of the ratio between the amplification products, as reported at the bottom of each gel. Furthermore, since the same competitor DNA fragment acts as a competitor for quantification of both CC–CKR-5 and β-actin, standardization for mRNA input is obtained by estimating the ratio between the two measurements, as indicated at the bottom of the figure. M, molecular weight markers.