IsolationofTotalRNAfromAnimalCellsuseRNeasyMiniKit

實驗概要

Extract the total RNA from animal cells by using RNeasy Mini Kit (QIAGEN No.74104)

主要試劑

SDS based extraction solution

實驗步驟

1.  Harvest cells.

1)   Trypsinize and collect cells, aspirate medium, and wash cells with PBS twice.

2)   Determine the number of cells.

3)    Transfer cells to an RNase-free centrifuge tube and pellet by  centrifugation at    300 x g for 5 min. (In our lab, use 1800rpm in the  eppendorf centrifuge 5415D). Cell pellets can be stored at –70°C for  later use or used directly in the procedure.

2.   Lyse cells

 1) Thaw frozen cell pellets slightly.

 2)  Add Buffer RLT (Add 10 μl –ME per 1 ml Buffer RLT). Usually, If number  of pelleted cells <5 x 106, add 350μl Buffer RLT.If number of  pelleted cells is  around 5 x 106 – 1 x 107, add 600μl Buffer RLT.

 3) Vortex or pipet to mix.

3.   Homogenize the sample.

       It is best to use a rotor–stator homogenizer to homogenize cells for 30  s. If not, pass the lysate at least 5 times through a needle fitted to  an RNase-free syringe.

4.   Add 1 volume (usually 350 μl or 600 μl) 70% ethanol, mix using pipet. Do not centrifuge.

5.    Take up to 700μl of that solution and run through a supplied mini  column for 15 seconds at 12,000 rpm. Repeat the previous step until all  of the solution has run through the mini column.

6.   Add 350 μl Buffer RW1 into the column, and centrifuge for 15 s at 12,000rpm to wash. Discard the flow-through.

7.    Add 10 μl DNase I stock solution (Dissolve the solid DNase I (1500  Kunitz units) in 550 μl of the RNase-free water.) to 70 μl Buffer RDD.  Mix gently.

8.   Add the DNase I incubation mix (80 μl) directly onto the RNeasy membrane, 20–30°C for 15 min.

9.   Run 700μl RW1 through the mini column, 15 seconds at 12,000 rpm.

10.   Change out the collection tube under the mini column. Run 500μl RPE  (ethanol added) through the column, 15 seconds at 12,000 rpm.

11.  Spin another 500μl RPE through the column for 2 minutes at maximum speed (13,200 rpm for us).

12.  Discard the flow-through. Spin another 1 minute at maximum speed (13,200 rpm for us) to ensure that the column is dry.

13.  Add 30-50μl RNase-free H₂O directly to the filter of the column, and spin for 1 minute at 12,000 rpm.

注意事項

1.        All steps performed at room temperature. 

2.        RNA can be stored at –80°C for short time.

3.        At step 8, it is needed to prolong the treated time for cell >5 x 106.