miRcute miRNA Isolation Kit

Protocol tips

Upstream tips	Protocol tips	Downstream tips
- Addition of Buffer MZ depends on monolayer area, not cell number as it will reflect on DNA contamination if not suffcient	- To capture more amount of mRNA, modify the amount of lysis buffer :ethanol (from 1:1 to 1:1.5) during the binding step.

Upstream tips
- Addition of Buffer MZ depends on monolayer area, not cell number as it will reflect on DNA contamination if not suffcient
Protocol tips
- To capture more amount of mRNA, modify the amount of lysis buffer :ethanol (from 1:1 to 1:1.5) during the binding step.

Publication protocol

Total RNA from human lung cancer cell lines was isolated from confluent cultures using the Absolutely RNA Microprep Kit (Stratagene, La Jolla, CA). 1 μg of total RNA was used in reverse transcriptase (RT) reactions using the superscript kit (Qiagen, Germantown, MD) and the RT reactions were subsequently used to set up real-time PCR reactions using1 μl of RT as a template. The real-time PCR reactions were set up using Syber green PCR master mix (BioRad, Hercules, CA) and the PCR reactions were run using the Opticon 2 PCR machine (BioRad). The PCR reactions were run using the following protocol: 1. incubate at 95°C for 10 min, 2. incubate at 95°C for 15 sec, 3. incubate for 30 sec, 4. go to line 2 for 39 more cycles, 8. melting curve from 60°C to 95°C, read every 1.0°C, 9. incubate at 10°C forever. The RT-PCR reactions were carried out in triplicate and the fold change was calculated using the 2T−ΔΔC method. Cyclophyllin was used as a loading control.

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Manufacturer protocol

Download the product protocol from Tiangen for miRcute miRNA Isolation Kit below.

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