RNAsimple Total RNA Kit

Protocol tips

Upstream tips	Protocol tips	Downstream tips
- To wipe off RNase, the glassware can be roasted at 150℃ for 4 hours, while plastic can be dipped in 0.5 M NaOH for 10min, washed by RNase-Free ddH2O thoroughly, and sterilized

Upstream tips
- To wipe off RNase, the glassware can be roasted at 150℃ for 4 hours, while plastic can be dipped in 0.5 M NaOH for 10min, washed by RNase-Free ddH2O thoroughly, and sterilized

Publication protocol

Total RNA in each sample was extracted by an RNA extraction kit (Tiangen Biotech Co., Ltd., Beijing, China) completely following the manufacturer instructions and reverse-transcribed into cDNA. RT-qPCR was carried out by Stratagene Mx3000P (Stratagene; Agilent Technologies, Inc., Santa Clara, CA, USA) using Bestar® SybrGreen qPCR MasterMix (DBI® Bioscience, Ludwigshafen, Germany) with the following protocol: Initial denaturation at 95°C for 2 min, 40 cycles consisting of 94°C for 20 sec, 58°C for 20 sec, and 72°C for 20 sec. The following primers were used: miR-126-5p, 5′-CTCAACTGGTGTCGTGGAGTCGGCAATTCAGTTGAGCGCGTA-3′ (sense) and 5′-ACACTCCAGCTGGGCATTATTACTTTTGGTA-3′ (antisense); VEGFR-A, 5′-AGGGCAGAATCATCACGAAGT-3′ (sense) and 5′-AGGGTCTCGATTGGAT-GGCA-3′ (antisense); VEGFR-2, 5′-ATAGAAGGTGCCCAGGAAAAG-3′ (sense) and 5′-GTCTTCAGTTCCCCTCCATTG-3′ (antisense); MEK1, 5′-GGGCTTCTA-TGGTGCGTTCTA-3′ (sense) and 5′-CCCACGGGAGTTGACTAGGAT-3′ (antisense); ERK, 5′-TCTGGAGCAGTATTACGACCC-3′ (sense) and 5′-CTGGCTGGAATCTAGCAGTCT-3′ (antisense); β-actin, 5′-ATCGTGCGTGACA-TTAAGGAGAAG-3′ (sense) and 5′-AGGAAGGAAGGCTGGAAGAGTG-3′ (antisense). Relative mRNA expressions were obtained by 2−∆∆CT method. β-actin was served as an internal control.

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Manufacturer protocol

Download the product protocol from Tiangen for RNAsimple Total RNA Kit below.

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