RNA extraction-PDF

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:

• RNA extraction using trizol/tri
• Trizol extraction for RNA and DNA

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)

• 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.