Clayton: We used Trizol from Invitrogen for that purpose and got pretty good quality and quantity of RNA. Check our paper (attached) for reference.

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Dear Clayton,

This is the method that I used for my own research.

RNA/mRNA isolation

This method is based on the fact that DNA is confined to the nucleus and RNA is found in the cytosol. Although this method is for extraction from the nuclei of the lymphocytes extracted from peripheral blood remain in tact, I feel confident that it can be adapted for other tissue cells. The RNA isolation from the cytosol is very similar to any other nucleotide extraction such as the isolation of DNA except negligible amounts of DNA is available in this case being confined to the unlysed nuclei.

Denaturing agents such as guanidine HCl and guanidinium thiocyanate (Cox 1968) readily dissolve proteins. Cellular structures disintegrate and nucleoproteins dissociate from nucleic acids as protein secondary structure is lost.

It is important to minimise the activity of RNases during cell lysis by using RNase inhibitors the cells are disrupted. Accidental contamination of trace amounts RNases from sources in the laboratory must also be avoided. The use of DEPC’ a strong inhibitor of RNase, in the preparation of glassware, plastic ware and electrophoresis tanks can prevent contamination.

RNA STAT-60 is composed of phenol and guanidinium thiocyanate in a monophase solution. Chloroform was added to the sample RNA STAT-60 mixture where the homogenate separated into two phases: aqueous phase and organic phase. RNA remains in the aqueous phase while the DNA and proteins are extracted in the organic phase and the interphase.  RNA was precipitated using isopropanol.

Reagents:

Chloroform (ACS grade)

Isopropanol (ACS grade)

Ethanol (ACS grade)

RNA STAT-60

Diethyl pyrocarbonate (DEPC) for ensuring RNase free environment.

Protocol:

RNA/mRNA isolation was performed using the RNA STAT-60 method in four stages

1.     Homogenization (RNA STAT-60 using 1ml/50-100mg of tissue or 5-10x106 cells)

2.     RNA extraction 1 volume of homogenate with 0.2 volumes of chloroform

3.     RNA precipitation using 0.5 volumes of isopropanol

4.     RNA wash using 75% ethanol

Unless otherwise stated all the procedures were performed at room temperature.

 Although long winded, this method is cheaper than some standard kits and reliable.

I hope that this is helpful and lays down some basic principles.

Best of luck,

Annwyne

Thank you everyone,

I have experience with Trizol. Just wanted to ensure I'd get good yield with brain tissue.

I really appreciate the advice.