RNA preparation from few worms

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The following protocol is suitable for RNA extraction from 5-10 adult worms or 20-30 larvae. I have successfully used it for single embryo RNA extractions/cDNA synthesis. In practice however this will not give amounts usable for qPCR on the LC480 cycler.

To extract RNA you will need the following reagents:

  • RNAse free water DEPC treated
  • Chloroform
  • Isopropanol
  • 70%EtOH
  • Trizol extracted, RNAse free Glycogen stocks at a concentration of 10 mg/ml
  • liquid Nitrogen
  • precooled Microcentrifuge
  • RNAse free eppendorf tubes
  • WORMS !
  • 1/10 dilution of Single Worm lysis buffer

The entire procedure takes about one hour in experienced hands:

  1. Pipet 10 µl of 1:10 diluted Single Worm lysis buffer into the lid of a microcentrifuge tube. I use 1:10 diluted single Worm lysis buffer simply because of its low surface tension - it evenly covers the inside of the lid and embryos/worms easily detach from the pick)
  2. Pick worms into the lid of the microcentrifuge tube; Eppendorf tubes are not very good for this step as every single tube has an inscription on the outer and a ridge of plastic on the inner side of the cap. I find Sarstedt tubes better because they are nicely flat and translucent. It is easier to see embryos detach from the pick in these tubes.
  3. Carefully close the tube and pulse spin to collect liquid at the bottom of the tube.
  4. Add 100 µl of Trizol, vortex briefly, pulse spin and let stand at RT for 10 minutes.
  5. Freeze tubes in liquid Nitrogen.
  6. Thaw tubes in 37° heat block.
  7. Repeat freeze-and-thaw 3 more times.
  8. Separate phases by adding 50 µl Chlorophorm, vortex 15 seconds and spin at 14 krpm for 15 minutes at 4°C.
  9. Transfer supernatant (and only supernatant, NO INTERPHASE OR TRIZOL) to a new RNAse free tube.
  10. Add 2 µl of Glycogen stock. Mix well.
  11. Precipitate RNA by adding 1 volume of Isopropanol.
  12. Pellet RNA by centrifugation at 14 krpm, 15 minutes, 4°C. You should see a small pellet (mostly consisting of glycogen) at the end of this step.
  13. Carefully take off supernatant and wash pellet with 100 µl of 70% EtOH.
  14. Centrifuge 5 minutes at 7500 rpm at 4°C.
  15. Take off EtOH and let RNA pellet dry for 5-10 minutes in the RNAse-free-hood.
  16. Resuspend pellet in 20 µl DEPC treated water and use as soon as possible. I usually directly do the RT reaction.

To do the first strand cDNA synthesis mix in a RNAse free tube:

  • 10µl RNA (from the above mentioned 20 µl resuspension volume)
  • 1µl 10mM dNTPS
  • 1µl dN6 random hexamers 100 mM
  • denature at 70 °C for 5 minutes
  • place on ice immediatley

Add:

  • 4 µl 5X buffer
  • 2 µl DTT 0.1 M
  • 1µl Supersript Reverse Transcriptase (Invitrogen 10 U / µl)
  • Always include a negative control without RT ! This is important as a negative control during the quantitative PCR.
  • Incubate at RT for 10 minutes
  • Incubate at 37°C in a PCR cycler for 1-2 hours followed by an obligatory heat-inactivation step at 72°C for 15 minutes. If you fail to heat inactivate the Reverse Transcriptase, residual activity will, while mixing the PCR reaction, ligate your primers and you will preferentially amplify Primer dimers during the QPCR.

Add 30 µl of DEPC treated water to the cDNA synthesis reaction. Use 5 µl of that as template in QPCR.

From here you may want to proceed with: Quantitative RT-PCR on Lighty cycler 480

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