Isolation of RNA from animal cells

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BACKGROUND

Isolation of total RNA from the animal cells is an essential step to initiate any experiment like cDNA synthesis, real-time PCR, RNA sequencing etc. When we isolate RNA is comes with all kind of RNAs from the animal cells. Trizol kit is very famous to apply for RNA isolation easy.

Aim of this experiment is to isolate all types of RNA from the animal cells.

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REQUIREMENTS

Sample: Animal cells

Apparatus: Cell scraper,

15 ml centrifuge tube,

1.5 ml Eppendorf tube,

Centrifuge machine.

Chemicals: Trizol kit,

1XPBS,

Chloroform,

Isopropyl alcohol,

70% ethanol in DEPC water,

DEPC water.

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PROCEDURE

Take a confluent animal cell plate, rinse the cells with PBS solution once and add 3-5 ml of PBS, scrap all cells in the PBS, and transfer it in 15 ml centrifuge tube. Centrifuge at 4000 rpm for 3-5 min and aspirate the supernatant further add 1 ml of PBS and mix it and transfer into an Eppendorf tube, centrifuge it with 12000 rpm for 2-3 min and aspirate the soup and add 1 ml Trizol and vertex it vigorously (5 min) and centrifuge it for 10 min at 12000 rpm. Collect aqueous phase (Fig.1) in a new tube, add chloroform (200 µl) vertex vigorously for 5-10 min and keep for 5 min at RT. Further centrifuge it at 12000 rpm for 10 min. Separate upper aqueous layer in new tube and add 500 µl isopropanol and flip-flop the tube keep in -20°C for at least 30 min further centrifuge at 12000 rpm for 10-15 min, discard supernatant and you can see the RNA pellet in the tube. Add 70% ethanol and centrifuge for 10 min at 12000 rpm. Keep open the tube for 10 min to dry the total RNA. Add 20 µl of DEPC water and store in -20°C for further applications.¹

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CONCLUSION

We concluded this experiment for the isolation of total RNA from the animal cells.

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REFERENCES

Kumar A, Huh TL, Choe J, Rhee M. Rnf152 Is Essential for NeuroD Expression and Delta-Notch Signaling in the Zebrafish Embryos. Molecules and Cells. 2017;40(12):945-53.