Jul 25, 2022
RNA extraction from diatom P. multistriata
- Francesco Manfellotto1,
- Antonella Ruggiero1,
- Pina Marotta1,
- Monia Teresa Russo1,
- Anna Santin1,
- Mariella Ferrante1
- 1Stazione Zoologica Anton Dohrn
Protocol Citation: Francesco Manfellotto, Antonella Ruggiero, Pina Marotta, Monia Teresa Russo, Anna Santin, Mariella Ferrante 2022. RNA extraction from diatom P. multistriata . protocols.io https://dx.doi.org/10.17504/protocols.io.261gen627g47/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it’s working
Created: December 20, 2021
Last Modified: July 25, 2022
Protocol Integer ID: 56178
Keywords: rna extraction, rna extraction protocol
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Abstract
RNA extraction protocol from diatom P. multistriata
Protocol materials
TRIzol ReagentThermo Fisher ScientificCatalog #15596026
Glass beads, acid-washed, 425-600 μm (30-40 U.S. sieve) Merck MilliporeSigma (Sigma-Aldrich)Catalog #G8772-100G
Troubleshooting
Collect 20/40 million cells
Harvest cells by filtration onto 1.2 µm pore size filter
Cut the filter into two halves and store each half in separate 2 mL eppendorf tube.
Add 1 mL of TRIzol® Reagent (for 5-10 × 10^6 cells) to eppendorf with half filter and vortex briefly.
TRIzol ReagentThermo Fisher ScientificCatalog #15596026
Flash freeze the eppendorf immediately in liquid nitrogen.
The eppendorf can be stored at -80 °C for later extraction.
Note: Do not wash cells before addition of TRIzol® Reagent.
Defrost the samples at room temperature (use thermoshaker for 15 sec at 60 °C if defrosting takes longer).
DO NOT PROCESS MORE THAN 6 SAMPLES AT SAME TIME.
Add 0,2g glass beads and put on thermoshaker at 60 °C for 10 min with maximum rpm (1400).Glass beads, acid-washed, 425-600 μm (30-40 U.S. sieve) Merck MilliporeSigma (Sigma-Aldrich)Catalog #G8772-100G
Centrifuge briefly for one minute to get rid of beads and filter and move the supernatant into 2 mL eppendorf tube.
Add 200 ul of CHLOROFORM per 1 ml of Trizol
Shake the eppendorf vigorously for 15 seconds
Incubate for 15 minutes at room temperature.
Centrifuge the eppendorf for 15 minutes at 10600 rpm (12,000 g) AT MAXIMUM , at 4 °C.
Transfer 500 ul (half the volume of Trizol used originally) of uppermost aqueous layer (contains RNA) into new 2mL eppendorf.
Do not disturb middle colorless phase and lowermost pink phase while pipetting (these phases contain proteins and DNA).
Add equal volume of ISOPROPANOL (500 ul) and invert the tube to mix.
Don’t shake.
Incubate at room temperature for 10 minutes
(samples can be stored at 4 °C overnight).
Centrifuge for 10 minutes at 10600 rpm at 4 °C.
Remove supernatant leaving RNA pellet undisturbed.
Add 1 mL of 75% ethanol. Invert the tube gently to wash the pellet
(samples can be stored at -20 °C for long time).
Centrifuge for 10 minutes at 8500 rpm (7500 g) at 4 °C.
Remove supernatant leaving RNA pellet undisturbed.
Allow the pellet to dry for 20-30 minutes to remove all the traces of ethanol.
Care should be taken not to over dry the pellet.
Re-suspend the pellet in 20-30 ul of RNA-se free water and dissolve the pellet by pipetting up and down.
For better re-suspending, incubate the samples for 5-10 minutes at 55-60 °C.