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[Arabidopsis] RNA extraction from dry seed

[Arabidopsis] RNA extraction from dry seed - Arabidopsis and Plant Biology

[Arabidopsis] RNA extraction from dry seed - Discuss Arabidopsis and Plant Biology Research.


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Old 02-04-2009, 07:26 PM
PEETAMBAR DAHAL
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Default [Arabidopsis] RNA extraction from dry seed



Seeds are special as these carry life to the next generation. So, we have to be extra careful to get RNA out of seeds. Below is our protocol.

RNA extraction protocol for seeds

1. Pulverize the seed tissue in LN. Add 7 mL Grinding buffer with 105 ul Beta-mercaptoethanol to 500 mg of seed tissue quickly (without thawing) and vortex well. Can add tiny bit of sea sand from Fisher to help grinding (too much will damage your probe)
2. Homogenize at the highest speed, 1 min each 3 times (Immerse the tube in ice bucket while homogenizing in the hood. Use double gloves, goggles). Pause 1 min between each grinding.
3. Shake the sample for 10-20 minutes at 300 rpm and centrifuge the sample for 20-30 minutes in table top-centrifuge or microplate centrifuge (RT, max speed ) or 6000-8000 g at 4.C (using swinging rotors only).
4. Collect the supernatant (Take out as much supernatant as possible without disturbing the interphase) and add equal volume of phenol : cholorform : isoamyl alcohol (25:24:1)—mix the solution by vortexing for 1 min—then spin for 20-30 min. (RT oK)
5. Collect the supernatant, note the volume, and add equal volume of 4 M LiCl for seeds (1/3 volume of 8M LiCl for other plant parts), mix thoroughly using clean side of parafilm and incubate the sample at –20C ON.
6. Spin 11,000 g for 30-40 min (4 C).
7. For carbohydrate contamination: Add 0.5 mL of 10 mM Tris pH 8.0 and 50 *l of 2M KOAc to the pellet. Incubate in ice for 20-30 min min. Spin 11,000 g for 10 min (4 C). Pipet out the supernatant and add 1.3 mL 100% ETOH to the supernatant in 2 mL eppies and incubate at -20 for ON or -80 C for at least 1 h.
8. Spin at 11,000 rpm (14,000Xg) at 4C and wash the pellet with 0.2-0.5mL 80% ETOH. (volume of ETOH should be adjusted based on pellet size). Spin @ 11,000 RPM for 5-10 min at 4 C. Dry the pellet for 20- 25 min at RT in laminar flow hood or use Vacuum for 5-7 min. (ETOH must be removed completely).
9. Dissolve in 50-200 ul DEPC treated water.

10. DNase treatment: (for RT and RT PCR):

Follow Turbo DNAse Free (including DNAse inactivating reagent) from Ambion.



Grinding Buffer = 15mL of TLE Buffer + 1.2mL of 2M NaOAc, pH 4.0, 15mL of phenol, 3mL chloroform.
TLE : 0.18 M Tris,, 0.09 M LiCl, 4.5 mM EDTA, 1% SDS, pH 8.2 with HCl (Current Protocols in Mol Biol, pp 4.3.3)
Phenol equilibration.: Melt phenol at 60 C. Transfer onto a clear DEPC treated 1 L bottle and wrap with aluminum foil. Add 0.1% 8-hydroxyquinoline and mix well (anti-oxidant as well as a coloring marker). Add equal volume of 1 M Tris (Make Tris in DEPC water and filter (0.22 *M) and mix thoroughly. Let it stand in cold and try to remove as much of I M Tris as possible. Then wash 3-4 times with 0.1 M pH 7.0 Tris similarly. (pH of the supn. is around 7.2).
pH meter: Immerse pH probe in 7 M GuHCl for 5 min and rinse the probe with room temperature DEPC treated water before checking pH of TLE buffer. Do not immerse the probe into phenol directly, rather check pH of supernatant. Any base or acid more than 1 M is regarded as RNAse free.

Notes:
I have scaled down the protocol for lettuce seeds and used 7 mL Grinding Buffer for 0.5 g seed in 15 mL polypropylene falcon tubes (do not use polystyrene tubes).

Peetambar Dahal

-----Original Message-----
From: [Only registered users see links. ] [mailto:[Only registered users see links. ].indiana.edu] On Behalf Of [Only registered users see links. ]
Sent: Tuesday, February 03, 2009 4:51 PM
To: Zacharie LeBlanc
Cc: [Only registered users see links. ]
Subject: Re: [Arabidopsis] RNA extraction from dry seed

Hi,

Most kits do not work with dry seeds, in my view.
I ever extracted total RNA from dry At seeds using the following
protocol and got RNA comparable to young tissue RNA extracted using
Qiagen Plant RNeasy mini kit. Good luck!

Suzuki Y, Kawazu T, Koyama H (2004) RNA isolation from siliques,
dry seeds, and other tissues of Arabidopsis thaliana. Biotechniques
37:542–544


Quoting "Zacharie LeBlanc" <[Only registered users see links. ]>:



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