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Table 1 Oligonucleotides and experimental parameters for QPCR, and oligonucleotides for in situ hydridization probe synthesis, for experiments in mouse fetal lungs

From: Expression of genes related to the hypothalamic-pituitary-adrenal axis in murine fetal lungs in late gestation

Genesa(NCBI)

Oligonucleotidesb

Amplicon length (bp)

QPCR conditionsc

 

QPCR: forward/reverse RNA probes: forward /reverse

Probe position

Ann. T°C

Acq. T°C

# of cycles

 

Target genes

Pomc (NM_008895)

CATTAGGCTTGGAGCAGGTC/TCTTGATGATGGCGTTCTTG

174

66

88

45

 

ACGTGGAAGATGCCGAGATTCTGCTACAGTC/CAGGAACAGCAGCAGTGC

141-433

   

Mc2r (NM_008560)

CCTCTCTTTGCTGGGCTCTA/GCACCCTTCATGTTGGTTCT

321

63

83

40

 

CCTCTCTTTGCTGGGCTCTA/GCACCCTTCATGTTGGTTCT

589-909

   

Crh (NM_205769)

CCTGGGGAATCTCAACAGAA/AACACGCGGAAAAAGTTAGC

120

62

89

45

 

TCCGCATGGGTGAAGAATAC/GGTGGAAGGTGAGATCCAGA

347-649

   

Crhbp (NM_198408)

TCTTACCAGAAGGAGCATCAG/GTGTCCGAGGGTAAGATCAG

241

63

81

45

 

CGGGGATTTCCTGAAGGTAT/CTGCAGTTTTGGTGCTGGTA

469-768

   

Crhr1 (NM_007762)

CCAGGATCAGCAGTGTGAGA/TGTTGTAGCGGACACCGTAG

161

64

86

43

 

CCAGGATCAGCAGTGTGAGA/AGTGGCCCAGGTAGTTGATG

265-372

   

Crhr2b (NM_009953)

CAGGCCAGGCACCCCAGGAC/GGAACCACGCGATGTTTCTCAG

477

68

85

43

 

GGCTTTACCTTGGTGGGTAG/TGAAAAATTCCCAGTTGTGGTC

162-492

   

Nr3c1 (NM_008173)

GGGGAATTCACAGACTTTCGGCTTCTGGA/GGGAAGCTTCTGACCTCCAAGGACTCTCG

297

63

83

40

 

ACCTGGATGACCAAATGACC/AGGAGAGAAGCAGTAAGGTTTTCA

1821-2078

   

Mrap (NM_029844)

TTCGTGGTGCTCCTCTTTCT/TCCTGGCTCCTCTGTTGTCT

162

63

85

43

Cyp21a1 (NM_009995)

CCTTGCCCCATCGTGCAACTA/TGGAGGCAGCAGAGTGAAGG

278

67

85

43

Vim (NM_011701)

GATCAGCTCACCAACGACAA/AAGACGTGCCAGAGAAGCAT

170

62

84

40

Krt18 (NM_010664)

GTGGAATCCAGACCGAGAAA/GTTCTTCCTTGAGTGCCTCG

406

58

84

40

 

Housekeeping genes

Gapdh (NM_008084)

GTCGGTGTGAACGGATTTG/AAGATGGTGATGGGCTTCC

215

61

84

40

Hprt1 (NM_013556)

AGTCCCAGCGTCGTGATTAG/AATCCAGCAGGTCAGCAAAG

228

64

82

40

Sdha (BC031849)

ACACAGACCTGGTGGAGACC/CAAACGGCTTCTTCTGCTGT

179

59

84

45

  1. aPomc, proopiomelanocortin; Mc2r, melanocortin receptor 2 (ACTH receptor); Crh, corticotropin releasing hormone; Crhbp, corticotropin releasing hormone binding protein; Crhr1, corticotropin releasing hormone receptor 1; Crhr2b, corticotropin releasing hormone receptor 2β; Nr3c1, nuclear receptor subfamily 3, group C, member 1 (glucocorticoid receptor); Mrap, melanocortin2 receptor accessory protein; Cyp21a1, 21-hydroxylase; Vim, vimentin; Krt18, keratin 18; Gapdh, glyceraldehyde-3-phosphate dehydrogenase; Hprt1, hypoxanthine phosphoribosyltransferase 1; Sdha, succinate dehydrogenase complex, subunit A, flavoprotein.
  2. bPrimer pairs used in QPCR experiments are presented. Primer pairs used to amplify and clone specific fragments to be used in RNA probe synthesis are underlined. Primer pairs for cloning contain sequences for restriction endonucleases: forward primers, GGGGAATTC; reverse primers, GGGAAGCTT.
  3. cAnn. T°C = annealing temperature; Acq. T°C = acquisition (fluorescence intensity reading) temperature.
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Reproductive Biology and Endocrinology

ISSN: 1477-7827

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