Eccles:RNA extraction AJ

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Two-step RNA extraction by using Tri Reagent and columns

Introduction

This is a tried and true protocol that is pretty much bullet-proof if performed as specified. The combination of RNA extraction from Tri Reagent (MRC) or Trizol (Invitrogen) homogenates combined with a column-based clean-up gives good quality RNA that is relatively free of inhibitors and works well for downstream application such as reverse transcription and RNA amplification for array work. We certainly have evidence that RNA isolated by this method works more reliably in downstream applications than Tri* alone.

General Guidelines

For this protocol we follow the first two step of the Tri* protocol - Homogenisation and Phase seperation - and then feed the resulting aqueous phase into a column-based clean-up. Both Invitrogen Purelink and Qiagen RNeasy column protocols include advice on how to to this, which is outlined here.

Materials Needed

• Tri Reagent (MRC; TR 118) or Trizol (Invitrogen; 15596-026).
• BCP (MRC; preferred) or chloroform.
• Columns, Invitrogen Purelink (12183-018) or Qiagen RNeasy (74104).
• 70% ethanol.
• Refridgerated centrifuge.

Procedure

Step 1 - Tri Reagent

• Tri Reagent protocol and Trizol protocol are essentially identical.
• The procedure is performed at room temperature,unless stated otherwise.
• Substituting BCP for chloroform does not affect the quality of isolated RNA, DNA or proteins and its use as the phase separation reagent may decrease the possiblity of contaminating RNA with DNA (4). Chloroform used for phase separation should not contain isoamyl alcohol or any other additive.
• It is important to perform centrifugation to separate aqueous and organic phases in the cold ( 4-10 dC ). If performed at elevated temperature, a residual amount of DNA may sequester in the aqueous phase. In this case, RNA can be used for northern analysis but it may not be suitable for PCR.

Homogenisation

• TISSUES. Homogenise tissue samples in TRI Reagent (1 ml/50 - 100 mg tissue) using a glass-Teflon or Polytron homogeniSer. Sample volume should not exceed 10% of the volume of TRI Reagent used for homogeniSation.
• CELLS. Cells grown in monolayer should be lysed directly in a culture dish. Pour off media, add TRI Reagent and pass the cell lysate several times through a pipette. Use 1 ml of TRI Reagnt per 10 cm^2 of culture dish area. See also note #3 in Notes to the RNA isolation protocol. Cells grown in suspension should be sedimented first, and then lysed in TRI REAGENT by repetitive pipetting. Use 1.0 ml of the reagent per 5 - 10 x 10^6 animal, plant or yeast cells or per 10^7 bacterial cells.
• Avoid washing cells before the addition of TRI Reagent as this may contribute to mRNA degradation. Disruption of some yeast and bacterial cells may require the use of a homogeniSer.

Phase seperation

1. Store the homogenate for 5 minutes at room temperature to permit the complete dissociation of nucleoprotein complexes.
2. Supplement the homogenate with 0.1 ml BCP or 0.2 ml chloroform per 1 ml of TRI Reagent, cover the samples tightly and shake vigorously for 15 seconds. DO NOT VORTEX.
3. Store the resulting mixture at room temperature for 2-15 minutes and centrifuge at 12,000 g for 15 minutes at 4 dC.
4. Following centrifugation, the mixture separates into a lower red phenol-chloroform phase, interphase, and the colourless upper aqueous phase. RNA remains exclusively in the aqueous phase whereas DNA and proteins are in the interphase and organic phase. The volume of the aqueous phase is about 60% of the volume of TRI Reagent used for homogenisation.
5. Transfer the aqueous phase to a fresh 1.5 mL tube and save the interphase and organic phase at 4 dC (-80 dC long-term) for subsequent isolation of DNA and proteins.

Step 2 - Column clean-up

Now we take the RNA-containing Tri* aqueous phase and pass it through a column. This second part of the protocol is performed exactly as laid out by the respective column manufacturers. The Purelink and RNeasy protocols are very similar - bind, wash, elute - but vary in the column spin speeds, so be careful not to mix up protocols from the different manufacturers. The following are lifted from the respective manufacturer's instructions.

Invitrogen Purelink

1. Add an equal volume of 70% ethanol to obtain a final ethanol concentration of 35% and mix well by vortexing. Mix thoroughly by inverting the tube. Disperse any visible precipitate that may form after adding ethanol. Proceed to Binding, Washing, and Elution on the next page.
2. Transfer up to 700 µl of the sample to the RNA Spin Cartridge pre-inserted in a collection tube. Centrifuge at 12,000 × g for 15 seconds at room temperature. Discard the flow-through, and re-insert the cartridge in the tube.
   • Repeat Steps 1-­2 until the entire sample has been processed.
   • Optional: If your downstream application requires DNA-free total RNA, you may use the convenient on-column DNase I treatment at this point in the procedure (see page 38).
3. Add 700 µl of Wash Buffer I to the spin cartridge. Centrifuge at 12,000 × g for 15 seconds at room temperature. Discard the flow-through and the collection tube.
4. Place the spin cartridge into a clean RNA Wash Tube, provided in the kit.
5. Add 500 µl of Wash Buffer II with ethanol (prepared as described on page 7) to the spin cartridge.
6. Centrifuge at 12,000 × g for 15 seconds at room temperature. Discard the flow-through, and re-insert the cartridge in the tube.
7. Repeat Steps 6-­7 once.
8. Centrifuge the spin cartridge at 12,000 × g for 1 minute at room temperature to dry the membrane with attached RNA.
9. Discard the collection tube, and insert the cartridge into an RNA Recovery Tube supplied with the kit.
10. To elute the RNA, add 30-­100 µl of RNase-free water to the center of the spin cartridge, and incubate at room temperature for 1 minute. See page 7 for detailed elution parameters (50 uL is my favourite volume, AJ).
11. Centrifuge the spin cartridge for 2 minutes at 12,000 × g at room temperature. If you are performing sequential elutions, collect all elutes into the same tube.
    • Determine the quantity and quality of the RNA by using the Agilent Bioanalyzer 2100 and Nanodrop ND-1000 spec. To perform DNase I digestion of the purified RNA, see page 39. If you will use the RNA within a few hours, store on ice. For longer storage, store at ­80°C. (I tend to give the eluant a good mix, spin dow, and then remove 3 uL - 1 uL for Nanodrop, 1 uL for Bioanalyzer, 1 uL for pipetting error - then store the remainder at -80dC, AJ).

Qiagen RNeasy

1. Add an equal volume of 70% ethanol to the aqueous phase obtained in 5, above, and mix immediately by pipetting up and down 5-10 times. Do not centrifuge. Proceed immediately to step 2. Note: Precipitates may be visible after addition of ethanol. This does not affect the procedure.
2. Transfer up to 700 µl of the sample, including any precipitate that may have formed, to an RNeasy spin column placed in a 2 ml collection tube (supplied). Close the lid gently, and centrifuge for 15 s at 8000 x g ( 10,000 rpm). Discard the flow-through. Reuse the collection tube in step 7. If the sample volume exceeds 700 µl, centrifuge successive aliquots in the same RNeasy spin column. Discard the flow-through after each centrifugation.
3. Add 700 µl Buffer RW1 to the RNeasy spin column. Close the lid gently, and centrifuge for 15 s at 8000 x g ( 10,000 rpm) to wash the spin column membrane. Discard the flow-through. Reuse the collection tube in step 8. Note: After centrifugation, carefully remove the RNeasy spin column from the collection tube so that the column does not contact the flow-through. Be sure to empty the collection tube completely.
4. Add 500 µl Buffer RPE to the RNeasy spin column. Close the lid gently, and centrifuge for 15 s at 8000 x g ( 10,000 rpm) to wash the spin column membrane. Discard the flow-through. Reuse the collection tube in step 9. Note: Buffer RPE is supplied as a concentrate. Ensure that ethanol is added to Buffer RPE before use (see "Things to do before starting").
5. Add 500 µl Buffer RPE to the RNeasy spin column. Close the lid gently, and centrifuge for 2 min at 8000 x g ( 10,000 rpm) to wash the spin column membrane. The long centrifugation dries the spin column membrane, ensuring that no ethanol is carried over during RNA elution. Residual ethanol may interfere with downstream reactions. Note: After centrifugation, carefully remove the RNeasy spin column from the collection tube so that the column does not contact the flow-through. Otherwise, carryover of ethanol will occur. Optional: Place the RNeasy spin column in a new 2 ml collection tube (supplied), and discard the old collection tube with the flow-through. Close the lid gently, and centrifuge at full speed for 1 min. Perform this step to eliminate any possible carryover of Buffer RPE, or if residual flow-through remains on the outside of the RNeasy spin column after step 9.
6. Place the RNeasy spin column in a new 1.5 ml collection tube (supplied). Add 30-­50 µl RNase-free water directly to the spin column membrane. Close the lid gently, and centrifuge for 1 min at 8000 x g ( 10,000 rpm) to elute the RNA. If the expected RNA yield is >30 µg, repeat step 11 using another 30-­50 µl RNase-free water, or using the eluate from step 11 (if high RNA concentration is required). Reuse the collection tube from step 11. If using the eluate from step 11, the RNA yield will be 15-­30% less than that obtained using a second volume of RNase-free water, but the final RNA concentration will be higher.