Eccles:RNA extraction AJ: Difference between revisions
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==General Guidelines== | ==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== | ==Materials Needed== | ||
*Tri Reagent or Trizol. | *Tri Reagent ([http://www.mrcgene.com/tri.html MRC]) or Trizol [https://catalog.invitrogen.com/index.cfm?fuseaction=viewCatalog.viewProductDetails&productDescription=189 Invitrogen]). | ||
*BCP (preferred) or chloroform. | *BCP (preferred) or chloroform. | ||
*Columns, Invitrogen Purelink or Qiagen RNeasy. | *Columns, Invitrogen Purelink or Qiagen RNeasy. | ||
| Line 16: | Line 16: | ||
==Procedure== | ==Procedure== | ||
===Tri Reagent=== | ===Tri Reagent=== | ||
The procedure is performed at room temperature,unless stated otherwise. | *Tri Reagent [http://www.mrcgene.com/tri.htm protocol] and Trizol ([http://www.invitrogen.com/content/sfs/manuals/15596026.pdf protocol] are essentially identical. | ||
*The procedure is performed at room temperature,unless stated otherwise. | |||
#HOMOGENIZATION | #HOMOGENIZATION | ||
#*TISSUES. Homogenize tissue samples in TRI Reagent (1 ml/50 - 100 mg tissue) using a glass-Teflon or Polytron homogenizer. Sample volume should not exceed 10% of the volume of TRI Reagent used for homogenization. | #*TISSUES. Homogenize tissue samples in TRI Reagent (1 ml/50 - 100 mg tissue) using a glass-Teflon or Polytron homogenizer. Sample volume should not exceed 10% of the volume of TRI Reagent used for homogenization. | ||
#*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 | #*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 homogenizer. | #*'''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 homogenizer. | ||
#PHASE SEPARATION | #PHASE SEPARATION | ||
#*Store the homogenate for 5 minutes at room temperature to permit the complete dissociation of nucleoprotein complexes. | #*Store the homogenate for 5 minutes at room temperature to permit the complete dissociation of nucleoprotein complexes. | ||
#*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. | #*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. | ||
#*Store the resulting mixture at room temperature for 2-15 minutes and centrifuge at 12,000 g for 15 minutes at 4 | #*Store the resulting mixture at room temperature for 2-15 minutes and centrifuge at 12,000 g for 15 minutes at 4 dC. | ||
#*Following centrifugation, the mixture separates into a lower red phenol-chloroform phase, interphase and the colorless 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 homogenization. | #*Following centrifugation, the mixture separates into a lower red phenol-chloroform phase, interphase and the colorless 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 homogenization. | ||
#*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. | #*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 | #*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. | ||
Revision as of 18:08, 29 May 2007
Back to Eccles Lab > DGG Protocols
RNA extraction by using Tri Reagent plus column clean-up
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) or Trizol Invitrogen).
- BCP (preferred) or chloroform.
- Columns, Invitrogen Purelink or Qiagen RNeasy.
- Refridgerated centrifuge.
Procedure
Tri Reagent
- Tri Reagent protocol and Trizol (protocol are essentially identical.
- The procedure is performed at room temperature,unless stated otherwise.
- HOMOGENIZATION
- TISSUES. Homogenize tissue samples in TRI Reagent (1 ml/50 - 100 mg tissue) using a glass-Teflon or Polytron homogenizer. Sample volume should not exceed 10% of the volume of TRI Reagent used for homogenization.
- 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 homogenizer.
- PHASE SEPARATION
- Store the homogenate for 5 minutes at room temperature to permit the complete dissociation of nucleoprotein complexes.
- 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.
- Store the resulting mixture at room temperature for 2-15 minutes and centrifuge at 12,000 g for 15 minutes at 4 dC.
- Following centrifugation, the mixture separates into a lower red phenol-chloroform phase, interphase and the colorless 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 homogenization.
- 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.
