Reverse transcription rt pcr kit

Total RNA was extracted from HeLa cells using TRIzol (Invitrogen, Carlsbad, CA, USA), and first strand cDNA reverse transcription was performed using a reverse-transcription RT-PCR kit (Takara) in the presence of Oligo (dT) 15 primers. qPCR was conducted using a LightCycler® 480 Real-Time PCR Detection System (Roche) and SYBR Green Real-Time PCR 2× premix kit (Takara). The reactions were performed under the following conditions: 15 sec at 95ºC followed by 40 cycles of denaturation for 15 sec at 95ºC and annealing and extension for 15 sec at 60ºC. The melting curves for Rab7, LC3 and p62 were determined and found to be specific. A two standard curve method was used to quantify the expression levels of Rab7, LC3 and p62, and the mRNA levels were normalized to that of β-actin. Primers used for Rab7, LC3, p62 and β-actin were as follows: Rab7 forward 5′-CATCCTGGGAGATTCTGGAGTC-3′ and reverse, 5′-TGT GTCCCATATCTGCATTGTG-3′; LC3 forward, 5′-GAGCAG CATCCAACCAAAATC-3′ and reverse, 5′-GCCTGATTA GCATTGAGCTGTAAG-3′; p62 forward, 5′-GGACTTGGT TGCCTTTTCCAGTG-3′ and reverse, 5′-GCAGCCGTC GCAGATCACAT-3′; β-actin forward, 5′-GCACCCAGC ACAATGAAGATC-3′ and reverse, 5′-CTCGTCATACTC CTGCTTGCTG-3′.