Polymerase chain reaction (PCR) primers were selected with the program Primer Quest (http://www.idtdna.com). Reverse primers were selected to be complementary to exon sequences within the human ENA-78, human GCP-2, or human glyceraldehyde-3-phosphate dehydrogenase (GAPD) coding regions. Forward primers for mRNA amplification spanned at least one exon–exon boundary, whereas primers for pre-mRNA amplification were selected to be complementary to sequences in an intron (forward primer), as previously described.
18 All primer sequences were analyzed by using BLAST (www.ncbi.nlm.nih.gov/blast/ provided in the public domain by the National Center for Biotechnology Information, Bethesda, MD), to ensure that the primers were specific and thus amplified only the gene of interest during PCR.
The primer sequences selected are listed, followed by the expected RT-PCR product size in parentheses: human ENA-78 primers (mRNA 493-bp product, pre-mRNA 275-bp product) mRNA forward oligonucleotide 5′-ATCTCCGCTCCTCCACCCAGT-3′, pre-mRNA forward oligonucleotide 5′-GCAATTACACACAGCTTGTCACT-3′, and mRNA and pre-mRNA reverse oligonucleotide 5′-TTCTTGTCTTCCCTGGGTTCAGA-3′; human GCP-2 primers (mRNA 179-bp product, pre-mRNA 333-bp product): mRNA forward oligonucleotide 5′-CTGGTCCTGTCTCTGCTGTGCT-3′, pre-mRNA forward oligonucleotide 5′-AGGGAACTCTCCCAGCAACCT-3′, mRNA and pre-mRNA reverse oligonucleotide 5′-GCTTCCGGGTCCAGACAAACT-3′; human GAPD primers (mRNA 417-bp product, pre-mRNA 286-bp product): mRNA forward oligonucleotide 5′-CCAAAGGGTCATCATCTCTGC-3′, pre-mRNA forward oligonucleotide 5′-AGAGGGGTGATGTGGGGAGTA-3′, mRNA and pre-mRNA reverse oligonucleotide 5′-ATTTGGCAGGTTTTTCTAGACGG-3′. The RT-PCR products were verified by size (agarose gel electrophoresis, described below) and sequence with an automated sequencer (model 373XL; Applied Biosystems, Inc., Foster City, CA).