Total RNA isolation from seedlings

[ampasaladr]

Please discuss about methods to isolate total RNA from seedlings

[superior]

Extraction Buffer (EB)
1M Tris-HCl pH9.0
1% SDS
autoclave
PCI
Phenol:Chloroform:Isoamyl alcohol (25:24:1)
The 24:1 chloroform:isoamyl alcohol can be prepared in advance and stored. PCI should be prepared fresh by mixing 1 part of TE-saturated phenol to 1 part of the 24:1 chloroform:isoamyl alcohol solution. This can be stored up to 1 week at room temp in the dark.
Also prepare:


*

3M NaOAc pH5.6 (autoclaved)
*

4M LiCl (autoclaved)
*

2M LiCl (autoclaved)
*

100% and 70% EtOH
*

mortar and pestles
*

13 eppen tubes/sample
*

ddH2O (autoclaved)

Protocol

* After harvesting, siliques should be immediately frozen in liquid N2. Samples are stored at -80°C until the extraction
* Place all solutions, eppen etc on ice to cool. Set centrifuge to 4°C. All steps are performed on ice and all centrifuge steps are at 4°C and 10,000 rpm
* All supernatant removal should also be carried out with a fine pippette. We use either a 200 ml yellow pippette tip attached to a pasteur pippette for this, or a fine pasteur pippette made using a bunsen burner

1.

Add 300µl of EB and 300 ml of PCI to an eppen tube on ice
2.

Cool the mortar & pestle with liquid N2, add 10 seed pods (or 0.05g of 8DAF siliques) per sample and homogenise very well, adding N2 as necessary to keep sample frozen
3.

Once fully ground, use small spoon to transfer sample to the eppen tube with the buffer
4.

vortex for 3 min and divide the sample into 2 eppen tubes
5.

Centrifuge for 1 min and collect sup into new eppen
6.

Add an equal volume of PCI and vortex
7.

Centrifuge for 5 min and collect sup into new eppen
8.

Centrifuge again for 5 min and collect sup into new eppen
9.

Add 0.1 vol 3M NaOAc and 3 vol 100% EtOH, invert 4 times to mix
10.

Place at -80°C (dry ice) for 10 min
11.

Centrifuge for 5 min, discard sup and vacuum dry pellet
12.

Dissolve pellet with 100µl ddH2O (may take time)
13.

Centrifuge for 10 min and collect sup into new eppen
14.

Add equal volume of 4M LiCl, invert 4 times to mix
15.

Place on ice on 4°C cold room overnight or -80°C (dry ice) for 1 hr

NEXT DAY

16.

Centrifuge for 15 min, discard sup. Small pellet should be visible, be careful as it moves easily
17.

Add 1 mL of 2M LiCl and carefully invert once to mix
18.

Centrifuge for 2 min and discard sup
19.

Add 1 mL of 70% EtOH and invert once to wash
20.

Centrifuge for 2 min, discard sup and vacuum dry
21.

Dissolve pellet in 5 to 10µl ddH2O
22.

Combine the 2 solutions/sample into one eppen
23.

Centrifuge for 5 min and collect sup into new tube
24.

Store at -30°C or lower
25.

Expected RNA amounts: 4DAF, 1-2µg; 6DAF, 2-3µg; 8DAF, 6-8µg; 10DAF, 8-10µg; 12DAF, 6-8µg; 14DAF, 3-4µg