Figure 4 of Weng, Mol Vis 2008; 14:562-571.


Figure 4. Reverse transcription polymerase chain reaction for detection of keratocan, keratin 12 and Cre mRNAs in testis. Total RNAs were isolated from four pooled corneas and individual testis of two Kera-Cre mice with TRIzol Reagent (Invitrogen), and then the total RNAs were dissolved in DEPC-Water and stored at −80 °C until use. Ten micrograms (testis) and 3.5 μg of total RNA were annealed to oligo-dT and reverse transcribed with kits from Promega according to the manufacturer’s instruction. Single stranded cDNA was subjected to PCR reactions using primer pairs for keratocan (Kera), keratin 12 (K12), Cre recombinase (Cre), and glyceraldehydes phosphate dehydrogenase (GAPDH) as described in Methods. A: Use of primers in exons 1 and 3 of Krt12 generates a 408 bp RT–PCR product with total RNA from the cornea, but not that from testis; B: use of primers in exons 7 and 8 of Krt12, a 410 bp RT–PCR product was detected with RNA from both corneas and testis; C: use of primers in exons 2 and 3 of Kera produces a 350 bp RT–PCR product with RNA from cornea and testis; D, Use primer of exon 1 of Kera and primer of IRES yields a 480 bp RT–PCR product in RNA from both cornea and testis of Kera-Cre mice; E: primers of exon 6 and 7 of GAPDH gene, a 200 bp transcript was used as a positive control RT–PCR with RNA from both cornea and testis. Lane 1, testis 1; lane 2, testis 2; lane 3, cornea; lane 4, control (no cDNA added); M, 1 kb DNA markers.