Eccles:RNA extraction AJ

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(Norgen Biotek Total RNA Purification Kit (Cat# 17200))
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*Norgen protocol available [http://www.norgenbiotek.com/display-product.php?ID=68  here].
*Norgen protocol available [http://www.norgenbiotek.com/display-product.php?ID=68  here].
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====Invitrogen Purelink====
====Invitrogen Purelink====
#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.
#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.
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#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.
#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.
#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.
#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.
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Revision as of 16:35, 2 August 2011

Back to Eccles Lab > DGG Protocols

Contents

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 steps 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 do 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.
  • Nuclease-free tips and tubes.
  • 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.
  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.

Norgen Biotek Total RNA Purification Kit (preferred column; cat# 17200)

  • The Norgen Total RNA kit is the preferred column as it retains all RNA sizes including miRNA below 200 nt; the Invitrogen and Qiagen Total RNA kits mentioned below do not retain small RNA. Tri Reagent feeds straight into the Norgen column protocol in the same way as for the other columns: mix equal volume of Tri aqueous phase with 70% EtOH, then onto column, and the column protocol.
  • Norgen protocol available here.


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