RNA extraction: Difference between revisions
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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). | #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.) | #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.) | ||
#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 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. 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. | |||
# | |||
==Related information== | ==Related information== | ||
Revision as of 09:58, 9 October 2007
This is a general protocol page for extracting RNA from cells.
Specific Protocols
- Endy:RNA extraction
- Extraction of RNA from tissues in English
- Extraction of RNA from tissues in French
- Gattuso:RNA extraction
Notes
Some general notes on isolating RNA.
These notes are focused on bacterial cells but please contribute to include information about plant, mammalian and other cells etc.
- 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.)
- 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. 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.
Related information
See RNA.
