求教RT-PCR
丁香园论坛
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我做大鼠肝组织TNFalpha受体的表达，用退火温度52，40个循环都做不出，为什么？它的表达量应该很丰富的呀，会不会RNA降解了，但内参跑出来了（240bp），是不是我从文献上查的引物有问题，请高手帮验证一下；
TNFR1 NM_013091
1 ttttctccga gttttctgaa ctctggctca tgatcgggct tactggatac gagaatcctg
61 gaggaccgta ccctgatttc catctacctc tgactttgag cctttctaac ccggggctca
121 cgctgccaac acccgggcca cctggtccga tcgtcttact tcattcacca gcgttgccaa
181 ttgctgccct gtccccagcc ccaatggggg agtgagagag gccactgccg gccggacatg
241 ggtctcccca tcgtgcctgg cctgctgctg tcactggtgc tcctggctct gctgatgggg
301 atacacccat caggggtcac cggactggtt ccttctcttg gtgaccggga gaagagggat
361 aatttgtgtc cccagggaaa gtatgcccat ccaaagaata attccatctg ctgcaccaag
421 tgccacaaag gaacctactt ggtgagtgac tgtccaagcc cagggcagga aacagtctgc
481 gaggtgtgtg ataaaggcac ctttacagct tcgcagaacc acgtcagaca gtgtctcagt
541 tgcaagacat gtcggaaaga aatgttccag gtggagattt ctccttgcaa agctgacatg
601 gacaccgtgt gtggctgcaa gaagaaccaa ttccagcgct acctgagtga gacgcatttc
661 cagtgtgtgg actgcagccc ctgcttcaat ggcaccgtga caatcccctg taaggagaaa
721 cagaacaccg tgtgtaactg ccacgcagga ttctttctaa gcggaaatga gtgcacccct
781 tgcagccact gcaagaaaaa tcaggaatgt atgaagctgt gcctacctcc agttgcaaat
841 gtcacaaacc cccaggactc aggtactgcc gtgctgttgc ctctggttat cttcctaggt
901 ctttgccttt tattctttat ctgcatcagt ctactgtgcc gatatcccca gtggaggccc
961 agggtctact ccatcatttg tagggattca gctcctgtca aagaggtgga gggtgaagga
1021 attgttacta agcccctaac tccagcctct atcccagcct tcagccccaa ccccggcttc
1081 aaccccactc tgggcttcag caccacccca cgcttcagtc atcctgtctc cagtaccccc
1141 atcagccccg tcttcggtcc tagtaactgg cacaacttcg tgccacctgt aagagaggtg
1201 gtcccaaccc agggtgctga ccctctcctc tacggatccc tcaaccctgt gccaatcccc
1261 gcccctgttc ggaaatggga agacgtcgtc gcggcccagc cacaacggct tgacactgca
1321 gaccctgcga tgctgtatgc tgtggtggat ggcgtgcctc cgacacgctg gaaggagttc
1381 atgcggctcc tggggctgag cgagcacgag atcgagcggc tggagctgca gaacgggcgt
1441 tgcctccgcg aggctcatta cagcatgctg gaagcctggc ggcgccgcac accgcgacac
1501 gaggccacgc tggacgtagt gggccgcgtg ctttgcgaca tgaacctgcg tggctgcctg
1561 gagaacatcc gcgagactct agaaagccct gcccactcgt ccacgaccca cctcccgcga
1621 taaggccaca cccccacctc aggaacggga ctcgaaggac catcctgcta gatgccctgc
1681 ttccctgtga acctcctctt tggtcctcta gggggcaggc tcgatctggc aggctcgatc
1741 tggcagccac ttccttggtg ctaccgactt ggtgtacata gcttttccca gctgccgagg
1801 acagcctgtg ccagccactt gtgcatggca gggaagtgtg ccatctgctc ccagacagct
1861 gagggtgcca aaagccagga gaggtgattg tggagaaaaa gcacaatcta tctgataccc
1921 acttgggatg caaggaccca aacaaagctt ctcagggcct cctcagttga tttctgggcc
1981 cttttcacag tagataaaac agtctttgta ttgattatat cacactaatg gatgaacggt
2041 tgaactccct aaggtagggg caagcacaga acagtggggt ctccagctgg agcccccgac
2101 tcttgtaaat acactaaaaa tctaaaagtg
引物： F 5’-TCTCAGTTGCAAGACATGTCG-3’
R 5’-TTGTGCCAGTTACTAGGACCG-3’（632bp）
TNFR1 NM_013091
1 ttttctccga gttttctgaa ctctggctca tgatcgggct tactggatac gagaatcctg
61 gaggaccgta ccctgatttc catctacctc tgactttgag cctttctaac ccggggctca
121 cgctgccaac acccgggcca cctggtccga tcgtcttact tcattcacca gcgttgccaa
181 ttgctgccct gtccccagcc ccaatggggg agtgagagag gccactgccg gccggacatg
241 ggtctcccca tcgtgcctgg cctgctgctg tcactggtgc tcctggctct gctgatgggg
301 atacacccat caggggtcac cggactggtt ccttctcttg gtgaccggga gaagagggat
361 aatttgtgtc cccagggaaa gtatgcccat ccaaagaata attccatctg ctgcaccaag
421 tgccacaaag gaacctactt ggtgagtgac tgtccaagcc cagggcagga aacagtctgc
481 gaggtgtgtg ataaaggcac ctttacagct tcgcagaacc acgtcagaca gtgtctcagt
541 tgcaagacat gtcggaaaga aatgttccag gtggagattt ctccttgcaa agctgacatg
601 gacaccgtgt gtggctgcaa gaagaaccaa ttccagcgct acctgagtga gacgcatttc
661 cagtgtgtgg actgcagccc ctgcttcaat ggcaccgtga caatcccctg taaggagaaa
721 cagaacaccg tgtgtaactg ccacgcagga ttctttctaa gcggaaatga gtgcacccct
781 tgcagccact gcaagaaaaa tcaggaatgt atgaagctgt gcctacctcc agttgcaaat
841 gtcacaaacc cccaggactc aggtactgcc gtgctgttgc ctctggttat cttcctaggt
901 ctttgccttt tattctttat ctgcatcagt ctactgtgcc gatatcccca gtggaggccc
961 agggtctact ccatcatttg tagggattca gctcctgtca aagaggtgga gggtgaagga
1021 attgttacta agcccctaac tccagcctct atcccagcct tcagccccaa ccccggcttc
1081 aaccccactc tgggcttcag caccacccca cgcttcagtc atcctgtctc cagtaccccc
1141 atcagccccg tcttcggtcc tagtaactgg cacaacttcg tgccacctgt aagagaggtg
1201 gtcccaaccc agggtgctga ccctctcctc tacggatccc tcaaccctgt gccaatcccc
1261 gcccctgttc ggaaatggga agacgtcgtc gcggcccagc cacaacggct tgacactgca
1321 gaccctgcga tgctgtatgc tgtggtggat ggcgtgcctc cgacacgctg gaaggagttc
1381 atgcggctcc tggggctgag cgagcacgag atcgagcggc tggagctgca gaacgggcgt
1441 tgcctccgcg aggctcatta cagcatgctg gaagcctggc ggcgccgcac accgcgacac
1501 gaggccacgc tggacgtagt gggccgcgtg ctttgcgaca tgaacctgcg tggctgcctg
1561 gagaacatcc gcgagactct agaaagccct gcccactcgt ccacgaccca cctcccgcga
1621 taaggccaca cccccacctc aggaacggga ctcgaaggac catcctgcta gatgccctgc
1681 ttccctgtga acctcctctt tggtcctcta gggggcaggc tcgatctggc aggctcgatc
1741 tggcagccac ttccttggtg ctaccgactt ggtgtacata gcttttccca gctgccgagg
1801 acagcctgtg ccagccactt gtgcatggca gggaagtgtg ccatctgctc ccagacagct
1861 gagggtgcca aaagccagga gaggtgattg tggagaaaaa gcacaatcta tctgataccc
1921 acttgggatg caaggaccca aacaaagctt ctcagggcct cctcagttga tttctgggcc
1981 cttttcacag tagataaaac agtctttgta ttgattatat cacactaatg gatgaacggt
2041 tgaactccct aaggtagggg caagcacaga acagtggggt ctccagctgg agcccccgac
2101 tcttgtaaat acactaaaaa tctaaaagtg
引物： F 5’-TCTCAGTTGCAAGACATGTCG-3’
R 5’-TTGTGCCAGTTACTAGGACCG-3’（632bp）
