User:Wilfred J. Poppinga/Notebook/cAMP compartmentalization/2010/06/08

 .todo { color: red } .done { color: green} {| width="800"
 * style="background-color: #EEE"|[[Image:owwnotebook_icon.png|128px]] Creating RNA isolation protocol
 * style="background-color: #F2F2F2" align="center"|  |Main project page
 * style="background-color: #F2F2F2" align="center"|  |Main project page


 * colspan="2"|
 * colspan="2"|

Summary
To obtain RNA for Real Time PCR RNA needs to isolated, to compare this to changes seen in protein expression. For best comparison also protein can be isolated from the same sample.

Materials

 * TRIzol® Reagent (invitrogen)
 * Chlorophorm
 * Isopropanol
 * RNeasy Mini kit (Qiagen)
 * RNase free water

Procedure

 * 1) Per Ø10 cm dish collect cells in 3 mL TRIzol® Reagent
 * 2) Incubate 5’ @ RT
 * 3) Add chloroform, 0.2 mL/mL TRIzol® Reagent
 * 4) Vortex to mix
 * 5) Incubate 2–3’ @ RT
 * 6) Centrifuge 15’ 12.000×g @ 4°C
 * 7) Collect supernatant
 * 8) Save red, phenol/chloroform phase for DNA/Protein isolation?
 * 9) Add isopropanol, 0.5 mL / mL TRIzol® Reagent
 * 10) Incubate 10’ @ RT
 * 11) Centrifuge 10’ 12.000×g @ 4°C
 * 12) Remove supernatant and resuspend pellet in 500 µL/ mL TRIzol® Reagent 100% EtOH
 * 13) Transfer sample (<700 µL) to RNeasy spin column (w. collection tube)
 * 14) Centrifuge 15 s, ≥8000×g (≥10.000 RPM), discard flowthrough
 * 15) >700 µL of sample can be put through the same column (<100 µg RNA)
 * 16) Add 700 µL of Buffer RW1 to column
 * 17) Centrifuge 15 s, ≥8000×g (≥10.000 RPM), discard flowthrough
 * 18) Add 500 µL of Buffer RPE to column
 * 19) Centrifuge 15 s, ≥8000×g (≥10.000 RPM), discard flowthrough
 * 20) Add 500 µL of Buffer RPE to column
 * 21) Centrifuge 2 min, ≥8000×g (≥10.000 RPM), discard flowthrough
 * 22) OPTIONAL, Centrifuge 1 min, ≥8000×g (≥10.000 RPM), discard flowthrough
 * 23) Put column to new 1.5 mL tube, add 30–50 µL RNAse free water
 * 24) Centrifuge 1 min, ≥8000×g (≥10.000 RPM)
 * 25) Determine RNA concentration by Nanodrop
 * 26) @ >10 µg RNA second elution using same column could be performed
 * 27) Store @ -20 or -70 °C

Extra material

 * 0.3 M guanidine hydrochloride (95.53 g/mol) in 95% EtOH
 * 0.28659 g / 10 mL 95% EtOH

Procedure

 * 1) Take the red, phenol/chloroform phase from step 7.1
 * 2) Precipitate DNA with 0.3 mL 100% EtOH per 1 mL TRIzol® Reagent
 * 3) Mix by inversion
 * 4) Incubate 2–3’ @ RT
 * 5) Centrifuge 15’ ≤2.000×g @ 4°C
 * 6) Collect supernatant (approx. 0.8 mL / mL TRIzol® Reagent)
 * 7) Pellet DNA can be used if desired for further processing (not included)
 * 8) Add isopropanol, 1.5 mL / mL TRIzol® Reagent
 * 9) Incubate 10’ @ RT
 * 10) Centrifuge 10’ 12.000×g @ 4°C
 * 11) Remove supernatant
 * 12) Add 2 mL 0.3 M guanidine hydrochloride in 95% EtOH / mL TRIzol® Reagent
 * 13) Incubate 20’ @ RT
 * 14) Centrifuge 5’ 7.500×g @ 4°C
 * 15) Remove supernatant
 * 16) Repeat step 35 – 38 up to a total of 3 times
 * 17) Add 2 mL of EtOH to the protein pellet
 * 18) Incubate 20’ @ RT
 * 19) Centrifuge 5’ 7.500×g @ 4°C
 * 20) Remove supernatant and dry pellet
 * 21) Resuspend pellet in 1% SDS solution (if necessary incubate @ 50°C to dissolve)
 * 22) Centrifuge 10’ 10.000×g @ 4°C
 * 23) Transfer supernatant to fresh tube
 * 24) Store @ -20°C

Attachment
   [[Media:RNA_isolation_for_Real_time_qPCR.doc|WORD Protocol]]


 * }