RNA extraction

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==Notes==
==Notes==
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Some general notes on isolating RNA.
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Some general notes on isolating RNA <cite>MeasuringGeneExpression</cite>.
These notes are focused on bacterial cells but please contribute to include information about plant, mammalian and other cells etc.
These notes are focused on bacterial cells but please contribute to include information about plant, mammalian and other cells etc.
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#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.
#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.)
#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.)
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#Homogenization can improve efficiency of RNA isolation.  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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#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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#Acid phenol extraction and alcohol precipitation is very cheap.  The drawbacks are that it is a long procedure, is prone to DNA contamination, can leave residual phenol in the sample inhibiting downstream reactions and doesn't inactivate RNases immediately.
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Revision as of 13:04, 9 October 2007

This is a general protocol page for extracting RNA from cells.

Contents

Specific Protocols

Notes

Some general notes on isolating RNA [1].

These notes are focused on bacterial cells but please contribute to include information about plant, mammalian and other cells etc.

  1. Centrifugation of cells can induce a stress response in cells altering transcript levels (potentially unequally across different transcripts).
  2. "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.
  3. 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).
  4. 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.)
  5. 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.
  6. 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.
  7. 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.)
  8. 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.
  9. Acid phenol extraction and alcohol precipitation is very cheap. The drawbacks are that it is a long procedure, is prone to DNA contamination, can leave residual phenol in the sample inhibiting downstream reactions and doesn't inactivate RNases immediately.

Related information

See RNA.

Reference

  1. isbn:0415374723. [MeasuringGeneExpression]
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