RNA EXTRACTION

This protocol details the methods used to extract RNA from cells, purify the RNA by a combination of phase extraction and chromatography, and prepare a labeled cDNA copy of the message fraction of the purified RNA. The protocol also describes the process of making fluorescent cDNA representations of the message pools within the isolated total RNA pools. This is accomplished by using the pure total RNA as a substrate for reverse transcription in the presence of nucleotides derivatized with either a Cy3 or a Cy5 fluorescent tag.

Materials

Trizol Reagent (#15596-018, Life Technologies, Rockville, MD)
RNeasy Maxi Kit (# 75162, Qiagen, Valencia, CA)
Chloroform
Ethanol (200 Proof USP Ethyl Alcohol)
DPBS (Dulbecco's phosphate buffered saline)
3M sodium acetate (pH 5.2)
dATP, dCTP, dGTP, dTTP, 100 mM each, store frozen, -20°C (#27-2035-02, Pharmacia, Peapack, NJ)
pd(T)12-18 resuspend at 1mg/ml, and store frozen -20°C ( #27-7858, Amersham Pharmacia Biotech)
Anchored oligo primer (anchored;5'-TTT TTT TTT TTT TTT TTT TTV N-3')
resuspend at 2mg/ml, store frozen -20°C (e.g. # 3597-006, Genosys)
CyTM3-dUTP, 1 mM, and CyTM5-dUTP, 1 mM, store -20°C, light sensitive
RNasinâ Rnase inhibitor, store -20°C (#N211A, Promega)
SUPERSCRIPTTM II Rnase H` Reverse Transcriptase Kit, store -20°C, (#18064-014, Life Technologies, Rockville, MD)
C0t-1 DNA, 1mg/ml, store frozen -20°C (#15279-011, Life Technologies, Rockville, MD)
0.5M EDTA( pH 8.0)
1 N NaOH
1M TRIS-HCL (pH7.5)
TE pH 7.4
DEPC water 50 X Tris Acetate Buffer
15 ml round bottom polypropylene centrifuge tubes
50 ml conical polypropylene centrifuge tubes
1.5 ml Eppendorf tubes
0.2 ml thinwall PCR tube
MicroCon 100 ( Amicon Cat No. 42412)
High speed centrifuge for 15 ml tubes
Clinical centrifuge with horizontal rotor for 50 ml conical tubes
Tissue homogenizer (e.g. Polytron PT1200 with Polytron-Aggregate-Dispergier-und-Mischtechnik 147a Ch6014 #027-30-520-0, Brinkmann Instruments Inc., Westbury, NY)

Reagents and Solutions

RPE Buffer

Add 4 volumes of ethanol per volume of RPE concentrate supplied in Quiagen Kit.

RW1 Buffer

Supplied in Qiagen Kit

75% EtOH

Ethanol (100%) 375 ml
DEPC H2O 125 ml

500 ml

10x low T dNTP Mix

5x First Strand Buffer

Provided with Superscript II

TAE Buffer

50X Tris Acetate Electrophoresis Buffer 20 ml
DEPC H2O 980 ml

1000 ml

Method

1. If starting with cells harvested from tissue culture, wash the cell pellet twice in DPBS.

2. If starting with cells from tissue culture, add 1 ml of Trizol per 2x107 cells and mix by shaking. If starting with tissue, add 100 mg of frozen tissue directly to 4 ml of Trizol, and dissociate by homogenization with a rotating blade tissue homogenizer.

3. Add 2/10 volume of chloroform and shake for 15 seconds.

4. Let stand for 3 minutes. Centrifuge at 12,000 x g for 15 minutes at 4°C.

5. Take off the supernatant and add it to a polypropylene tube, recording the volume of the supernatant.

6. Add 0.53 volumes of ethanol to the supernatant slowly while vortexing, this will produce a final ethanol concentration of 35%.

The ethanol should be added drop by drop and allowed to mix completely with the supernatant before more ethanol is added. If a high local concentration of ethanol is produced, the RNA in that vicinity will precipitate.

7. Add the supernatant from an extraction of 2x107 to 1x108 cells to an RNeasy maxi column, which is seated in a 50 ml centrifuge tube.

8. Centrifuge at 2880 x g in a clinical centrifuge with a horizontal rotor at room temperature for 5 minutes.

9. Pour the flow-through back onto the top of the column and centrifuge again.

A significant amount of RNA is not captured by the column matrix in the first pass of the RNA containing solution through the column.

10. Discard the flow-through and add 15 ml of RW1 buffer to the column.

11. Centrifuge at 2880 x g for 5 minutes.

12. Discard flow-through then add 10 ml of RPE buffer.

13. Centrifuge at 2880 x g for 5 minutes.

14. Discard flow-through and add another 10 ml of RPE buffer.

15. Centrifuge at 2880 x g for 10 minutes.

16. Put the column in a fresh 50 ml tube and add 1 ml of DEPC treated water from the kit to the column.

17. Let stand for 1 minute.

18. Centrifuge at 2880 x g for 5 minutes.

19. Add another 1 ml of water to the column.

20. Let stand for 1 minute.

21. Centrifuge at 2880 x g for 10 minutes.

22. Aliquot out 400 µl portions of the column eluate to 1.5 ml Eppendorf tubes.

23. Add 1/10 volume of 3M sodium acetate (pH 5.2).

24. Add 1 ml of ethanol to each tube.

25. Let stand for 15 minutes.

26. Centrifuge at 12000 x g at 4C for 15 minutes.

27. Wash pellet 2 times in 75% EtOH then store at -80°C

RNA Cleanup

28. Resuspend RNA at approximately 1 mg/ml in DEPC H2O.

29. Concentrate to greater than 7 mg/ml by centrifugation on a MicroCon 100 filter unit, centrifuge at 500 x g, checking as necessary to determine the rate of concentration.

This step removes many residual, small to medium sized, molecules that inhibit the reverse transcription reaction in the presence of fluorescently derivatized nucleotides.

30. Determine the concentration of RNA in the concentrated sample by spectrophotometry. Store at -80°C.