LIFE09200604007 Tabish - Homi Bhabha National Institute
LIFE09200604007 Tabish - Homi Bhabha National Institute
LIFE09200604007 Tabish - Homi Bhabha National Institute
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Materials and Methods<br />
7. Ethidium bromide (10mg/ml)<br />
Method:<br />
A 1.2% reducing agarose gel was prepared by melting 0.36 gm agarose in 25.5<br />
ml DEPC water and cooled down to 55 °C. To this 3 ml 10x MOPS, 1.5 ml<br />
formaldehyde was added and the gel was allowed to set at RT. 1µl of RNA was mixed<br />
with above mentioned RNA loading buffer and heated at 65 °C for 7 min. sample was<br />
allowed to cool and after adding 2 µl of loading dye and 0.2 µl EtBr samples were<br />
loaded on the gel and run at a constant voltage of 50 V for 1.5 h. RNA bands were<br />
visualized and captured on UV exposure on a Gel documentation system.<br />
3.4.3.3 DNase treatment of RNA:<br />
Any downstream application of RNA requires it to be free of DNA<br />
contamination hence β-actin PCR was performed on isolated RNA to ensure any DNA<br />
content and samples were treated with DNase using DNA-free kit wherever required.<br />
Materials:<br />
1. DNA free kit (Ambion)<br />
2. Total RNA<br />
3. Water bath<br />
Method:<br />
For DNase treatment RNA concentration should be ≤200 ng/µl. Total RNA<br />
with ≈200 ng/µl concentration was taken in thin walled 0.6 ml eppendorf tube.<br />
Depending upon the amount of RNA being treated with the enzyme, per 10 µl reaction,<br />
1 µl 10x DNase Buffer and 1 U γ DNase I enzyme was added and the reaction was<br />
incubated at 37 °C for 30 min. To stop the enzyme activity 1 µl DNase inactivator<br />
slurry was added and the reaction was incubated for 2 min at RT with intermittent<br />
mixing so that inactivator can interact with total enzyme in the reaction mixture. The<br />
contents were centrifuged at 10,000 rpm/ RT/ 1.5 min and RNA was transferred to<br />
fresh tube taking care not to pick up the slurry.<br />
3.4.3.4 Preparation of cDNA from total RNA:<br />
cDNA is synthesized in vitro from a RNA template using reverse<br />
transcriptase. This process is called reverse transcription (RT) or first strand cDNA<br />
synthesis. The purpose of converting mRNA to cDNA is mainly for the analysis of the<br />
template mRNA because DNA is much stable than RNA. Once mRNA is converted to<br />
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