Note: Precautions should be taken to avoid ribonuclease contamination during the isolation procedure. Wear latex gloves or equivalent at all times when handling kit components. Any glassware used should be treated prior to its use (3). All kit components have been treated to ensure that they are ribonuclease free.
Additional Components Required:
- Allow oligo (dT)25-cellulose, column(s) and buffers to come to room temperature.
- Prepare a 65–70°C bath and an ice bath.
- Spin tube containing oligo (dT)25-cellulose in a microcentrifuge for 10 seconds. Using a micropipette remove storage buffer. Be careful to avoid drawing of cellulose beads into pipette tip.
- Equilibrate cellulose by adding 200 μl of Wash Buffer to cellulose beads, mix thoroughly then microcentrifuge for 10 seconds. Using a micropipette decant supernant.
- Prewarm Elution Buffer in 70°C bath.
- Ethanol (95%)
- Sterile 13 x 100 mm disposable test tubes
- Note: It is recommended that microcentrifuge be set at 2,000 to 5,000 x g. Do not exceed 12,000 x g.
- Rotary or other type of bidirectional shaker
- Isolation Procedure:
Add 50 μl of 5M NaCl per 450 μl of cell lysate or total RNA solution or dissolve total RNA sample in 450 μl of Elution Buffer then add 50 μl of 5M NaCl. Make sure that sample is totally dissolved. If not, microcentrifuge for 5 minutes to pellet insoluble material. Transfer RNA solution to clean microcentrifuge tube.
- Heat at 65°C for 5 minutes and quickly cool in an ice bath for 3 minutes. For information regarding the preparation of total RNA from eukaryotic cells or tissue see Maniatis, T., et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor, second edition, pp. 7.6–7.25 (1989).
- Apply total RNA solution to equilibrated oligo (dT)25 -cellulose, seal cap and mix thoroughly. Let stand at room temperature for 5 minutes agitating by hand or place horizontally on rotary shaker.
- Microcentrifuge for 10 seconds.
Note: It is important to agitate beads during binding, washing and elution steps.
- Pipet supernatant back into original microcentrifuge tube. Repeat steps 2 thru 4.
- Pipet supernatant back into original microcentrifuge tube for storage.
It is recommended that no spin-column eluates be discarded until entire isolation procedure is completed and the results are evaluated. Eluates can be stored in sterile test tubes on ice.
- Add 400 μl of Wash Buffer to oligo (dT)25 -cellulose beads. Agitate by hand to resuspend the cellulose beads. Using a 1 ml micropipette with sterile pipette tip, transfer Wash Buffer and beads to the column reservoir of a clean microcentrifuge spin column unit (provided with kit).
- Let stand at room temperature for 2 minutes agitating by hand or place horizontally on rotary shaker. Microcentrifuge for 10 seconds. Remove column reservoir and transfer column eluent to a clean 13 x 100 mm test tube.
- Add 400 μl of Wash Buffer to column reservoir and wash as in Step 8 three times.
- Using the same method wash column once with 400 μl of Low Salt Buffer.
*Note: This wash step with 0.1 M NaCl removes residual poly(A)– RNA which is bound to the cellulose column. This step can be omitted during a second round purification.
- Remove spin-column reservoir and place in a clean microcentrifuge tube (provided with kit).
- Add 200 μl of prewarmed Elution Buffer to column reservoir. Agitate by hand resuspending the cellulose beads. Let stand for 2 minutes agitating by hand or place on rotary shaker. Microcentrifuge for 10 seconds.
- Repeat Step 12 using fresh prewarmed Elution Buffer.
- Place Elution Buffer eluate on ice.
At this point quantification of isolated poly(A)+ can be done by spectrophotometric measurement at A260 nm. This is most easily done in a 0.5 ml quartz cuvette.
Soak cuvette for at least one hour in methanol: concentrated HCl (1:1), then rinse copiously with H2O which has been DEPC-treated.
For RNA 1 A260 U = approx. 40 μg
If second round purification is to be done immediately, equilibrate fresh oligo (dT)25 -cellulose beads and repeat isolation procedure starting at step 1. If poly(A)+ eluate is to be used at this point it can be ethanol precipitated:
a) To the poly(A)+ eluent add 44 μl of 3 M Sodium Acetate, 20 μl of Glycogen Carrier and 1.0 ml of cold 95% ethanol. Allow to stand at -20°C for at least 30 minutes.
At this point poly(A)+ material can be stored as an ethanol precipitate at -70°C until it is needed.
To recover poly(A)+ material microcentrifuge for 15 minutes at 4°C. Carefully decant supernant, then wash the pellet (often not visible) with 70% ethanol. Recentrifuge briefly, decant supernant and allow pellet to air dry.
Resuspend poly(A)+ material as required.