RNA extraction

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cmc, adapted from hkeller
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[[Image:Hub icon.png|right|protocol hub]]
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==Prepare RNase-Diminished bench==
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This is a general protocol page for extracting RNA from cells. The page also links to individual RNA extraction protocols (see at the end). Some sections link to separate pages with more in-depth explanations (see main page..).
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#Wipe down thoroughly with EtOH
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#Use Rnase Zap wipes to clean bench and pipettes
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#Wipe all down with a wet (H20) paper towel (2x).
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==Culture growth and sample collection==
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==Comparison of purification approaches==
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#Samples from continuous culture in chemostat:
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-
##[[Chemostat SOC]]
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##At desired time, measure OD600 of chemostat culture
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##Pull 2mL sample from chemostat through bubbler line. Immediately proceed to cell collection.
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#Samples from batch culture:
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##Inoculate 5mL standard media (see protocol, M9_recipe) from fresh plate (<2 weeks). Grow to saturation at 37° C, approx 20 hr.
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##Measure OD600 of saturated cultures.  All should be ~2.5.
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##Dilute all saturated cultures to OD600=0.0025 (dilution ~1:1000) into 5mL fresh media. (5μL culture if OD600=2.5).
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##Grow to mid-log, OD600 ~ 0.4
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##Pull 2mL sample and proceed immediately to cell collection.
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==Cell collection==
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===Guanidine isothiocyanate extraction coupled with lithium chloride precipitation===
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#Immediately combine 0.8mL cold stop solution with each 1mL sample in a pre-chilled centrifuge tube. (Stop solution= 5% H2O saturated phenol in ethanol).
+
*Lyses cells, extracts cellular RNA and denatures proteins all at once in many cases.
-
#Store on ice while plating dilutions of culture for CFU count. Plate three replicates of a 1:104 and a 1:105 dilution.
+
*Inactivates RNases faster than acid phenol extraction.
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#Centrifuge samples at 4°C in Sauer Lab tabletop centrifuge. 13,000rpm, 10 minutes.
+
*Does not physically separate RNA from proteins and DNA in one step. Protein contamination must be removed by chloroform treatment and need to differentiate between RNA and DNA using some other technique (like lithium chloride precipitation).
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#Decant the supernatants from the pellets and discard.
+
*Can also use cesium chloride ultra-centrifugation instead of lithium chloride precipitation but this requires access to an ultra-centrifuge.
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#''(optional)'' At this point, the pellets can be frozen at -80°C until ready to proceed with step 4.  Thaw on ice before lysis.
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==Lysis==
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===Acid phenol extraction and alcohol precipitation===
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#Resuspend each pellet in 0.5 ml of Lysis buffer (2% SDS and 4 mM EDTA).
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*Very cheap
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#Incubate the cells in a 100°C sand bath for 3 minutes to lyse the cells.
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*Long procedure
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#Add 29.3 μl of 3M NaOAc to each tube and transfer to ice.
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*Prone to DNA contamination
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#''(optional)'' spike in [[RNA In Vitro Standards|control mRNA]] to evaluate the efficiency of extraction
+
*Can leave residual phenol in the sample inhibiting downstream reactions and introducing error into RNA quantitation.
 +
*Doesn't inactivate RNases immediately.
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==Phenol Extraction with heat==
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===TRIzol or tri followed by chloroform and precipitation ===
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#Add an equal volume (0.5 mL) of water-saturated phenol to each tube.
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* trizol or tri (name depends on manufacturer) combines phenol and guanidine isothiocyanate and thereby some of the advantages of the above two
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#Invert several times to mix.
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* removes protein and DNA but depends on pipetting skills (disturbing the phases leads to contamination)  
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#Transfer to a 67°C water bath for 6 minutes and invert every 40 seconds.
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* RNA is protected by the reagent during the extraction procedure
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#Immediately transfer to ice.
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* phenol and chloroform are potentially harmful reagents (handle under the hood)
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#Centrifuge in an Eppendorf 5414 table top microcentrifuge (15,000 RPM) at 4°C for 10 minutes.
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#Transfer as much of each aqueous layer as possible to new tubes.  The aqueous layer is the upper layer and is distinct from the organic phase which is tinted yellow by 8-Quinolinol in the phenol
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==Phenol/Chloroform Extraction==
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For detailed protocols see:
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#Add an equal volume (0.5 mL) of water-saturated phenol:chloroform:isolamyl alcohol (25:24:1) to each tube.
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* [[RNA extraction using trizol/tri]]
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#Invert several times to mix.
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* [[Trizol extraction for RNA and DNA]]
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#Centrifuge in an Eppendorf 5414 table top microcentrifuge (15,000 RPM) at 4°C for 5 minutes.
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#Transfer as much of each aqueous layer as possible to new tubes.
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#Repeat phenol/chloroform extraction once.
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==Ethanol Precipitation==
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===Anion-exchange matrices===
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#To each tube, add: 1/10 volume (50μl) 3M NaOAc, 1/10 volume (50 μl) 1 mM EDTA, and 2-2.5 volumes (1 mL) of cold, 100% ethanol.
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*Allows purification of genomic DNA and RNA in parallel.
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#Invert to mix and then incubate at -80°C for 20 minutes.
+
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#Centrifuge in an Eppendorf 5414 table top microcentrifuge (15,000 RPM) at 4°C for 25 minutes.
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#Decant the ethanol from the pellets and discard.
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#Wash each pellet in 1 ml of cold 80% ethanol.
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#Centrifuge in an Eppendorf 5414 table top microcentrifuge (15,000 RPM) at 4°C for 5 minutes.
+
-
#Decant the ethanol from the pellets and discard. 
+
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#Repeat wash with cold 80% EtOH twice, for a total of three washes.
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#Dry the pellets in a 37°C heat block after the final wash is removed, ~15 min
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#Resuspend each pellet in 50 μl of Buffer EB.
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#''(Optional)'' Pool replicates of each sample in order to increase the total cellular RNA per sample (helpful for Northern Blot or RPA, not necessary for RT-PCR.)
+
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==Absorbance reading==
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===Silica matrices===
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*[[Nanodrop]] (make sure to set for RNA). Record concentration and A260/A280 ratio. An ideal purity has an A260/A280 between 1.8 and 2.2.
+
*Very small species (< 200nt) do not bind to silica matrices (except for Norgen columns which get miRNAs).
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==Storage==
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===Commercially available kits===
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*If the sample is to be treated immediately with DNase, proceed to the next section. Otherwise, dilute and/or aliquot the samples if necessary and store at
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* Qiagen RNeasy kits (silica column tubes) are the most widely recommended.  It is more expensive than other kits but is good for transcriptomics.
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-20°C until needed.
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* The column binding system of RNAeasy is faster and without harmful chemical but it has a lower maximum yield (due to the binding capacity of the columns) and will remove RNA smaller than ~200 nt.
 +
* All the kits are suitable for applications like RT-PCR as long as you check for and remove contaminants from RNA preparation.
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==DNase treatment==
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==Harvesting cells==
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#Combine:
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*Centrifugation of cells can induce a stress response in cells altering transcript levels (potentially unequally across different transcripts).
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#*50 μL of 10x DNase I Buffer
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*[http://www1.qiagen.com/literature/handbooks/literature.aspx?id=1000278 "RNA''protect''"] is a reagent used to stabilize RNA content in bacterial cells grown in liquid culture.
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#*50 – 100 L of RNA Extraction product
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**Works best if cells are grown in minimal media.  Works worst in LB liquid culture due to complex components.
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#*2 μL of Superase Inhibitor
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*Prepare all reagents and organize all equipment ahead of time to minimize the time between end of cell growth and cell lysis (especially if stabilization reagents are not used).
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#*5 μL DNase I (~1 ug enzyme)
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*Once cells are lysed, keep all samples on ice and use ice cold reagents to reduce RNase activity. However, note that RNases are still active at 0&deg;C. (Do not put cells on ice prior to cell lysis otherwise you risk inducing a cold-shock response.)
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#*H2O (RNase-free) to total volume of 500 μL
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#Incubate 10 min at 37°C.
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#''Optional:'' If using NEB DNase I, heat inactivate 10 min at 75°C.
+
-
==Phenol/Chloroform Extraction==
+
==Lysing cells==
-
#Add an equal volume (0.5 mL) of water-saturated phenol:chloroform:isolamyl alcohol (25:24:1) to each tube.
+
-
#Invert several times to mix.
+
-
#Centrifuge in an Eppendorf 5414 table top microcentrifuge (15,000 RPM) at 4°C for 5 minutes.
+
-
#Transfer as much of each aqueous layer as possible to new tubes.
+
-
==Ethanol Precipitation==
+
*Cell lysis is the point at which things are most likely to go wrong.
-
#To each tube, add: 1/10 volume (50μl) 3M NaOAc, 1/10 volume (50 μl) 1 mM EDTA, and 2-2.5 volumes (1 mL) of cold, 100% ethanol.
+
*Mechanical disruption and homogenization can help with cell lysis issues. Using a bead grinding machine may be helpful.
-
# Invert to mix and then incubate at -80°C for 20 minutes.
+
-
#Centrifuge in an Eppendorf 5414 table top microcentrifuge (15,000 RPM) at 4°C for 25 minutes.
+
-
#Decant the ethanol from the pellets and discard.
+
-
#Wash each pellet in 1 ml of cold 80% ethanol.
+
-
#Centrifuge in an Eppendorf 5414 table top microcentrifuge (15,000 RPM) at 4°C for 5 minutes.
+
-
#Decant the ethanol from the pellets and discard. 
+
-
#Repeat wash with cold 80% EtOH twice, for a total of three washes.
+
-
#Dry the pellets in a 37°C heat block after the final wash is removed, ~15 min.
+
-
#Resuspend each pellet in 30 μl of nuclease free water if to be used in RPA or in 10 μL Buffer EB if to be used in Northern Blot.
+
-
==Absorbance reading==
+
===Bacteria===
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*[[Nanodrop]] (make sure to set for RNA). Record concentration and A260/A280 ratio. An ideal purity has an A260/A280 between 1.8 and 2.2.
+
*The best way to lyse bacterial cells is guanidine isothiocyanate which isolates cellular RNA at the same time.
 +
**Pre-digestion of the cell wall may improve lysis efficiency.  This is essential for Gram-positive bacteria.  Use 0.4mg/mL lysozyme for Gram-negative bacteria and 3mg/mL lysozyme for Gram-positive bacteria in chosen resuspension buffer.
 +
*Use 2mL of lysozyme containing buffer per 10mL of E. coli bacterial culture having an OD600nm of 0.5 (assumes OD600nm  of 1 equals 10<sup>9</sup> cells per mL). Scale the amount of resuspension buffer linearly based on the number of cells to be lysed.
 +
*To lyse cells, add resuspension buffer and pipette up and down rapidly using a 250 &mu;L capacity pipette tip until no cell clumps are visible. Incubate on the bench for 5 mins (Gram-negative bacteria) or 15 mins (Gram-positive bacteria).  (This lysis procedure assumes that RNA stabilization reagents have been used or that resuspension buffer contains RNase inhibitors.)
 +
*Homogenization can improve efficiency of RNA isolation from bacterial cells.  If cell lysate is viscous, pour lysate into a syringe and pass the solution back and forth through a 20 gauge needle 5-10 times.
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==Storage==
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==RNA separation from other cellular molecules ==
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*Store at -20°C until needed.
+
Several different possibilities exist to purify RNA from complete cell lysate (see the comparison above). For mammalian cell culture and tissues trizol/tri and silica columns (e.g. RNAeasy) are probably the most common.
-
[[Category:RNA Protocol]]
+
 
 +
{{Main|RNA extraction using trizol/tri}}
 +
 
 +
== Solubilization of RNA ==
 +
* To dissolve RNA pellets, use RNase-free water (which is least likely to interfere with downstream applications).
 +
* If required heat the RNA After adding water to promote solubilization (70&deg;C for 5 mins, mix by pipetting every minute). 
 +
* RNA at a concentration of 1 &mu;g/&mu;L is too dilute to re-precipitate upon cooling.
 +
 
 +
== Quantification ==
 +
RNA is typically quantified spectrometrically using absorption at 260nm (A<sub>260</sub>). An A<sub>260</sub> reading of 1 is equivalent to ~40 µg/ml of single-stranded RNA [http://www.protocol-online.org/prot/Protocols/RNA-Quantitation-and-Purity-Determination-3538.html]. Machines that require very little sample volume for testing like NanoDrop are popular.
 +
 
 +
* Absorption measurement depends on the buffer (use the same buffer as blank)
 +
 
 +
{| {{table}}
 +
|-
 +
! style="background:lightgrey"|diluent
 +
! style="background:lightgrey"|A260/A280
 +
|-
 +
|DEPC water (pH 5-6)
 +
|1.60
 +
|-
 +
|Nuclease-free water (pH 6-7)
 +
|1.85
 +
|-
 +
|TE (pH 8.0)
 +
|2.14
 +
|-
 +
|}
 +
 
 +
* Depending on machine the A<sub>260</sub> reading of the RNA sample should be between 0.1-0.5.  Dilute the sample in H<sub>2</sub>O for this measurement.
 +
* NanoDrop's upper limit for RNA concentration is 3000 µg/µl; dilute if necessary
 +
* absorption is '''highly pH dependent''' (see also table above and PMID 9067025)
 +
 
 +
== Storage ==
 +
 
 +
*Store RNA at less than -70&deg;C. (why?)
 +
*Only freeze-thaw RNA samples once. (freeze thaw as little as possible)
 +
*Re-determine RNA concentration following storage and defrosting.
 +
 
 +
==Sources of contamination==
 +
====DNA====
 +
*To identify low levels of DNA contamination, do a PCR of a housekeeping gene and a portion of the RNA preparation as template.  If there is contamination, there will be products in all samples.
 +
*Use lithium chloride re-precipitation to remove DNA for best results.  However, this is slow.
 +
*Use DNaseI if you are in a hurry.
 +
====Protein====
 +
*Measure th A<sub>260</sub>/'''A<sub>280</sub>''' ratio.  It should be '''2.0''' for very pure RNA samples.  When measuring this ratio, dilute the RNA sample in TE buffer (not H<sub>2</sub>O) because pH can affect the A<sub>280</sub> reading.
 +
*If there is protein contamination, you may need to do a chloroform cleaning and reprecipitation.
 +
====Salt====
 +
*Measure the A<sub>260</sub>/'''A<sub>240</sub>''' ratio.  It should be '''1.4''' for very pure RNA samples. 
 +
*Isopropanol precipitation coupled with a 70% v/v ethanol wash of the pellet can remove salts.
 +
 
 +
== Appendix ==
 +
 
 +
=== See also ===
 +
* [[RNA]]
 +
 
 +
===Specific Protocols===
 +
*[[Endy:RNA extraction]]
 +
*[[Etchevers:RNA tissues|Extraction of RNA from tissues in English]]
 +
*[[Etchevers:Extraction ARN francais|Extraction of RNA from tissues in French]]
 +
*[[Gattuso:RNA extraction]]
 +
 
 +
===Reference===
 +
<biblio>
 +
#MeasuringGeneExpression isbn=0415374723
 +
</biblio>
 +
 
 +
=== External links ===
 +
* [http://www.ambion.com/techlib/tn/94/949.html Ambion tips on RNA quantification]
 +
 
 +
 
 +
[[Category:Protocol]]
 +
[[Category:RNA]]
 +
[[Category:In vitro]]

Current revision

protocol hub

This is a general protocol page for extracting RNA from cells. The page also links to individual RNA extraction protocols (see at the end). Some sections link to separate pages with more in-depth explanations (see main page..).

Contents

Comparison of purification approaches

Guanidine isothiocyanate extraction coupled with lithium chloride precipitation

  • Lyses cells, extracts cellular RNA and denatures proteins all at once in many cases.
  • Inactivates RNases faster than acid phenol extraction.
  • Does not physically separate RNA from proteins and DNA in one step. Protein contamination must be removed by chloroform treatment and need to differentiate between RNA and DNA using some other technique (like lithium chloride precipitation).
  • Can also use cesium chloride ultra-centrifugation instead of lithium chloride precipitation but this requires access to an ultra-centrifuge.

Acid phenol extraction and alcohol precipitation

  • Very cheap
  • Long procedure
  • Prone to DNA contamination
  • Can leave residual phenol in the sample inhibiting downstream reactions and introducing error into RNA quantitation.
  • Doesn't inactivate RNases immediately.

TRIzol or tri followed by chloroform and precipitation

  • trizol or tri (name depends on manufacturer) combines phenol and guanidine isothiocyanate and thereby some of the advantages of the above two
  • removes protein and DNA but depends on pipetting skills (disturbing the phases leads to contamination)
  • RNA is protected by the reagent during the extraction procedure
  • phenol and chloroform are potentially harmful reagents (handle under the hood)

For detailed protocols see:

Anion-exchange matrices

  • Allows purification of genomic DNA and RNA in parallel.

Silica matrices

  • Very small species (< 200nt) do not bind to silica matrices (except for Norgen columns which get miRNAs).

Commercially available kits

  • Qiagen RNeasy kits (silica column tubes) are the most widely recommended. It is more expensive than other kits but is good for transcriptomics.
  • The column binding system of RNAeasy is faster and without harmful chemical but it has a lower maximum yield (due to the binding capacity of the columns) and will remove RNA smaller than ~200 nt.
  • All the kits are suitable for applications like RT-PCR as long as you check for and remove contaminants from RNA preparation.

Harvesting cells

  • Centrifugation of cells can induce a stress response in cells altering transcript levels (potentially unequally across different transcripts).
  • "RNAprotect" is a reagent used to stabilize RNA content in bacterial cells grown in liquid culture.
    • Works best if cells are grown in minimal media. Works worst in LB liquid culture due to complex components.
  • Prepare all reagents and organize all equipment ahead of time to minimize the time between end of cell growth and cell lysis (especially if stabilization reagents are not used).
  • Once cells are lysed, keep all samples on ice and use ice cold reagents to reduce RNase activity. However, note that RNases are still active at 0°C. (Do not put cells on ice prior to cell lysis otherwise you risk inducing a cold-shock response.)

Lysing cells

  • Cell lysis is the point at which things are most likely to go wrong.
  • Mechanical disruption and homogenization can help with cell lysis issues. Using a bead grinding machine may be helpful.

Bacteria

  • The best way to lyse bacterial cells is guanidine isothiocyanate which isolates cellular RNA at the same time.
    • Pre-digestion of the cell wall may improve lysis efficiency. This is essential for Gram-positive bacteria. Use 0.4mg/mL lysozyme for Gram-negative bacteria and 3mg/mL lysozyme for Gram-positive bacteria in chosen resuspension buffer.
  • Use 2mL of lysozyme containing buffer per 10mL of E. coli bacterial culture having an OD600nm of 0.5 (assumes OD600nm of 1 equals 109 cells per mL). Scale the amount of resuspension buffer linearly based on the number of cells to be lysed.
  • To lyse cells, add resuspension buffer and pipette up and down rapidly using a 250 μL capacity pipette tip until no cell clumps are visible. Incubate on the bench for 5 mins (Gram-negative bacteria) or 15 mins (Gram-positive bacteria). (This lysis procedure assumes that RNA stabilization reagents have been used or that resuspension buffer contains RNase inhibitors.)
  • Homogenization can improve efficiency of RNA isolation from bacterial cells. If cell lysate is viscous, pour lysate into a syringe and pass the solution back and forth through a 20 gauge needle 5-10 times.

RNA separation from other cellular molecules

Several different possibilities exist to purify RNA from complete cell lysate (see the comparison above). For mammalian cell culture and tissues trizol/tri and silica columns (e.g. RNAeasy) are probably the most common.

Main article: RNA extraction using trizol/tri

Solubilization of RNA

  • To dissolve RNA pellets, use RNase-free water (which is least likely to interfere with downstream applications).
  • If required heat the RNA After adding water to promote solubilization (70°C for 5 mins, mix by pipetting every minute).
  • RNA at a concentration of 1 μg/μL is too dilute to re-precipitate upon cooling.

Quantification

RNA is typically quantified spectrometrically using absorption at 260nm (A260). An A260 reading of 1 is equivalent to ~40 µg/ml of single-stranded RNA [1]. Machines that require very little sample volume for testing like NanoDrop are popular.

  • Absorption measurement depends on the buffer (use the same buffer as blank)
diluent A260/A280
DEPC water (pH 5-6) 1.60
Nuclease-free water (pH 6-7) 1.85
TE (pH 8.0) 2.14
  • Depending on machine the A260 reading of the RNA sample should be between 0.1-0.5. Dilute the sample in H2O for this measurement.
  • NanoDrop's upper limit for RNA concentration is 3000 µg/µl; dilute if necessary
  • absorption is highly pH dependent (see also table above and PMID 9067025)

Storage

  • Store RNA at less than -70°C. (why?)
  • Only freeze-thaw RNA samples once. (freeze thaw as little as possible)
  • Re-determine RNA concentration following storage and defrosting.

Sources of contamination

DNA

  • To identify low levels of DNA contamination, do a PCR of a housekeeping gene and a portion of the RNA preparation as template. If there is contamination, there will be products in all samples.
  • Use lithium chloride re-precipitation to remove DNA for best results. However, this is slow.
  • Use DNaseI if you are in a hurry.

Protein

  • Measure th A260/A280 ratio. It should be 2.0 for very pure RNA samples. When measuring this ratio, dilute the RNA sample in TE buffer (not H2O) because pH can affect the A280 reading.
  • If there is protein contamination, you may need to do a chloroform cleaning and reprecipitation.

Salt

  • Measure the A260/A240 ratio. It should be 1.4 for very pure RNA samples.
  • Isopropanol precipitation coupled with a 70% v/v ethanol wash of the pellet can remove salts.

Appendix

See also

Specific Protocols

Reference

  1. Matthew B. Avison. Measuring gene expression. New York, NY: Taylor & Francis Group, 2007. isbn:0415374723. [MeasuringGeneExpression]

External links

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