Amv reverse transcriptase kit

Total RNA in cell lines was isolated using RNAiso Plus RNA Kit (TaKaRa, Dalian, People’s Republic of China) according to the manufacturer’s instructions. In order to determine the effect of siNEDD9, total RNA was isolated at 24, 48, and 72 hours after cell transfection. Quantitation of total RNAs was performed by spectrophotometry (UNICO, Shanghai, People’s Republic of China). All samples had an excellent 260/280 ratio. RNAs were reverse transcribed into complementary DNAs (cDNAs) by using the AMV reverse transcriptase kit (TaKaRa) and were stored at −20°C for immediate or later use.
The products of qRT-PCR were electrophoresed in 1% agarose gel and detected using a Gel-doc2000 gel scan imaging analysis system. Relative expression of NEDD9 mRNA was determined according to the ratio of NEDD9 and β-actin density. The expression of β-actin was assayed for normalization of mRNA expression.
For the mRNA expression of NEDD9 and β-actin, a 50 μL reaction containing 5 μL of 10× PCR buffer, 1 μL of 10 mM deoxynucleotide mixture, 0.5 μL of Taq enzyme, 2 μL of NEDD9 or β-actin primer, 36.5 μL RNase-free water, 3 μL of 25 mM magnesium chloride, and 2 μL template cDNA. Reaction mixtures were incubated under the following conditions: 94°C for 5 minutes, 20 cycles at 94°C for 30 seconds, 62°C for 30 seconds, 72°C for 45 seconds, and finally, 72°C for 7 minutes.

Total RNA was extracted using an RNA Extraction kit (Bioteck, Beijing, China). Single strand cDNA was reversely transcribed from 500 to 1000 ng RNA by using oligo dT-Adaptor primers and AMV reverse transcriptase kit (TaKaRa, Otsu, Japan). cDNA was detected using quantitative real-time PCR assay with a SYBR Green RealMasterMix kit (Tiangen, Beijing, China). GAPDH was used as an endogenous ‘housekeeping’ gene to normalize RNA levels across samples. The primer sequences for genes of interest and controls were listed in supporting table.