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Table I

Artificial RNA Standards and Primers for Quantitative RT-PCR

mRNAStandard sequenceUpper primerLower primerBufferReference
Albumin  391-584; NcoI (215)  391-407  568-584   Minghetti et al., 1985  
Transferrin  750 bp of 3′ end in vector  5′-TCA ACC TCA  5′-GCA GCG AAG   Ruppert et al., 1990  
   pmTF-2; NcoI   CGA CTC CT-3′   ACT ACA CC-3′     
Keratin 8  1299-3′-end; d1317 1348  1299-1316  1439-1456   Morita et al., 1988  
Keratin 18  904-3′-end; d921-952  904-920  1023-1040   Singer et al., 1986  
HNF-1  1122-1997; AocI (1715)  1494-1511  1765-1782   Kuo et al., 1990  
β-Globin  pGEM3Zf (1357-1550)  38390-38407  39436-39453   Shehee et al., 1989  
ε-Globin  pGEM3Zf (1357-1550)  9625-9642  10482-10499   Shehee et al., 1989  
Erythropoietin  1239-3201; AccI (2365)  1303-1320  3149-3166   Shoemaker et al., 1986 
c-jun  1430-1492/811 bp neo/2205-2304  1430-1447  2287-2304   Ryder and Nathans, 1988  
junB  1081-1483; d1249-1332  1081-1098  1466-1483   Ryder et al., 1988  
junD  pGEM3Zf (1132-1275)  519-535  732-748   Ryder et al., 1989  
c-fos  2969-3574; AccI (3220)  2969-2992  3551-3574   van Beveren et al., 1983  
fosB  6379-6830; d6418-6592  6397-6414  6813-6830   Lazo et al., 1992  
c-myc  969-1407; d1003-1186  969-986  1390-1407   Stanton et al., 1984  
Cyclin D2  55-520; d228-293  55-76  502-520   Matsushime et al., 1991  
Hepatocyte growth factor  1186-1748; d1384-1595  1186-1203  1731-1748   Liu et al., 1993  
Glyceraldehyde-3-phosphate  326-1152; CelII (510)  428-445  787-804   Sabath et al., 1990  
 dehydrogenase           
mRNAStandard sequenceUpper primerLower primerBufferReference
Albumin  391-584; NcoI (215)  391-407  568-584   Minghetti et al., 1985  
Transferrin  750 bp of 3′ end in vector  5′-TCA ACC TCA  5′-GCA GCG AAG   Ruppert et al., 1990  
   pmTF-2; NcoI   CGA CTC CT-3′   ACT ACA CC-3′     
Keratin 8  1299-3′-end; d1317 1348  1299-1316  1439-1456   Morita et al., 1988  
Keratin 18  904-3′-end; d921-952  904-920  1023-1040   Singer et al., 1986  
HNF-1  1122-1997; AocI (1715)  1494-1511  1765-1782   Kuo et al., 1990  
β-Globin  pGEM3Zf (1357-1550)  38390-38407  39436-39453   Shehee et al., 1989  
ε-Globin  pGEM3Zf (1357-1550)  9625-9642  10482-10499   Shehee et al., 1989  
Erythropoietin  1239-3201; AccI (2365)  1303-1320  3149-3166   Shoemaker et al., 1986 
c-jun  1430-1492/811 bp neo/2205-2304  1430-1447  2287-2304   Ryder and Nathans, 1988  
junB  1081-1483; d1249-1332  1081-1098  1466-1483   Ryder et al., 1988  
junD  pGEM3Zf (1132-1275)  519-535  732-748   Ryder et al., 1989  
c-fos  2969-3574; AccI (3220)  2969-2992  3551-3574   van Beveren et al., 1983  
fosB  6379-6830; d6418-6592  6397-6414  6813-6830   Lazo et al., 1992  
c-myc  969-1407; d1003-1186  969-986  1390-1407   Stanton et al., 1984  
Cyclin D2  55-520; d228-293  55-76  502-520   Matsushime et al., 1991  
Hepatocyte growth factor  1186-1748; d1384-1595  1186-1203  1731-1748   Liu et al., 1993  
Glyceraldehyde-3-phosphate  326-1152; CelII (510)  428-445  787-804   Sabath et al., 1990  
 dehydrogenase           

Bp numbering corresponds to the published cDNA or genomic DNA sequences. Restriction enzymes used for generation of standards with a mutated restriction site, the positions of the deletions in deletion standards (d) and the positions of the heterologous sequences on plasmid pGEM3z, amplified for heterologous standards, are indicated. In the case of the heterologous standard for c-jun, a part of the c-jun sequence between bp 1492 and 2205 is replaced by the neomycin resistance gene sequence of transposon TN5 (Hilberg et al., 1993). The indicated buffers correspond to the various PCR buffers of the PCR optimizer kit (Invitrogen).  

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