RNA Extraction from INS-1 cells using TriZol.

1) Obtain dishes with INS-1 cells that have reached confluence >80% of cell surface and drain off all media.

2) Wash with sterile PBS (optional).

3) In RNAse free hood, wash each dish with TriZol. Approximately 1mL of trizol per dish can be utilized to prepare cells for ‘scraping’. Using cell

4) Add 0.2 ml of chloroform per every 1.0 mL of TriZol. (eg: 1.6 mL of chloroform can be added to every 8 mL of TriZol utilized). After addition of chloroform, shake tube vigorously.

5) Allow incubation for 2-3 minutes at 15-30 C

6) Centrifuge at 12000g for 15 mins at 2-8C (or centrifuge at 3200g for 30 minutes at room temp)

7) Centrifugation will separate the different products into 2 layers. Extract the topmost aqueous layer (primarily containing RNA) and transfer into 1.5 mL centrifuge tubes (eppendorf). Add 1mL of the aquaeous layer to each of the centrifuge tubes.

8) Add 0.5 mL of isopropyl alcohol to each centrifuge tube.

9) Incubate samples at room temperature for 10 minutes. z

10) Centrifuge at 12000g for 10 mins at 2-8C (or centrifuge at 3200g for 30 minutes at room temp)

11) Extract and discard supernatant

12) Wash RNA pellet with 75% ethanol, adding atleast 1 mL of 75% ethanol per 1 mL of TriZol.

13) Mix sample by vortexing and centrifuge at 3200g for