Deoxynucleoside triphosphate

Total RNA was isolated from cells or tissues using TRIzol reagent (Invitrogen; Thermo Fisher Scientific, Inc., Cergy Pontoise, France). Next, 3 mg total RNA was denatured for 10 min at 70°C and then reversed transcribed into cDNA at 37°C for 90 min using 300 U Moloney murine leukemia virus reverse transcriptase, 15 mg oligo dT primers (both Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, USA) and 1 mM deoxynucleoside triphosphate (Bioline, London, UK) in a total volume of 30 ml. qPCR was then performed using a SYBR Green PCR Master Mix kit (ABgene; Thermo Fisher Scientific, Inc., Courtaboeuf Cedex, France) supplemented with 0.5 mM primers. The PCR mixture contained 7.5 µl SYBR Green, 4.5 µl water, 1 µl forward and reverse primers, respectively, and 2 µl DNA template. The primers were: Human TMEM35 forward, 5′-TGGGGACTATCAAGCTGACC-3′, and reverse, 5′-CAATGCTTTTTCGGAGGAGA-3′; β-actin forward, 5′-AATCGTGCGTGACATTAAGGAG-3′, and reverse, 5′-ACTGTGTTGGCGTACAGGTCTT-3′. The thermal cycling conditions used were as follows: 95°C for 15 min, then 40 cycles at 95°C for 20 sec, 58°C for 15 sec, and 72°C for 15 sec. Signals with a threshold cycle (Cq) value of >39 were considered to indicate no transcription of the target gene. The relative expression of mRNA was calculated using the 2^−ΔΔCq method (25 (link)).