Rnase free dnase 1 set

RNA was prepared using the RNeasy kit (Qiagen Inc., Valencia, CA) and was treated with DNase to remove contaminating genomic DNA, using RNase-free DNaseI set (Qiagen Inc., Valencia, CA). A total of 900 ng of RNA was reverse-transcribed with random hexamers in a 40 μL reaction using SuperScript III (Invitrogen Corp, Carlsbad, CA). Primers were synthesized for MISR2 and GapDh by SA Bioscience (Qiagen, MD), and primers for Alk2 (forward: 5′-CAGCTTGATGTGGGGAAC-3′ and reverse: 5′-GACT CGAGCGGCCGCTCAACAGTCAC-3′), Alk3 (forward: 5′-GCTCTATTTGATTACTGATTACCATG-3′ and reverse: 5′-TCCACCGATTAGACACAATTGGC-3′) and Alk6 (forward: 5′-CTCAGGGAGCGACCTGGGCA-3′ and reverse: 5′-GCGGCCCCAAATGCAGGGAT-3′) were synthesized by IDT (Coralville, Iowa). PCR was performed using 25-μl volume, containing 200 ng of cDNA, 1 unit of AmpliTaq DNA polymerase, PCR buffer, dNTPs (Applied Biosystems, Foster City, CA) and 10 pmole/μl primers, with appropriate controls. The cDNA was amplified in Bio-Rad iCycler using the following protocol: 95°C for 3 min; 35 cycles of 95°C for 30 s, 50°C for 30 s, and 72°C for 45 s; with a final step at 72°C for 7 min. PCR product of Alk2 (450bp), Alk3 (520bp), Alk6 (540bp), and MISR2 (120bp) were detected using 2% agarose gel. Gapdh was used as a housekeeping gene. Bands were visualized using G:BOX gel imager from Syngene (Frederick, MD).