Wednesday, June 03, 2009

how to extract total RNA from tissue samples


After I posted a method for extracting RNA from tissue samples, which someone had given me, I have spent the last few weeks composing an improved version which should be suitable for extracting total RNA from tissue. The difference is that the first method is really only suitable for extracting RNA for use in Quantitative (or real-time) PCR where you are looking to quantify a specific mRNA. This latter version is what I will be using for generating a normalised cDNA library by transcriptomic analysis, which requires complete extraction of the undegraded transcriptome. RNA is very vulnerable to degradation by RNase enzymes which comprise one of the many regulatory pathways in gene expression. Therefore you must be uber-keen to avoid any degradation. Transcriptomic analysis and the preparative steps involved in generating normalised cDNA libraries s also very sensitive to contamination and so the extraction must be as pure as possible.


RNA extraction from animal tissue for the creation of normalised cDNA libraries.

This method requires an RNeasy mini kit from Qiagen as well as an RNase-free DNase kit.

Pre-extraction prep:

• Prepare DNase I stock solution before using the RNase-Free DNase.
• Dissolve the lyophilized DNase I (1500 Kunitz units) in 550 μl of the Rnasefree water provided.
• Take great care not to lose any of the lyophilised powder.
• Mix gently by inverting the vial. ~~ Do not vortex! ~~
• Aliquot to 60µl volumes and store at -20°C

• Add 4 volumes of ethanol (96–100%) to the Buffer RPE concentrate, as indicated on the bottle to obtain a working solution.
• Aliquot to 1300µl volumes and store at -20°C

• Prepare some 70% EtOH

• Aliquot buffer RW1 into 1850µl volumes and freeze @ -20°C

• Aliquot buffer RDD into 400µl volumes and store @ -20°C

• Aliquot RNase-free water into 200µl volumes and freeze @ -20°C

Extraction protocol for 5 samples:

You will need:
• 70% EtOH
• 2 x buffer RW1 aliquots
• 1 x buffer RDD aliquot
• 2 x buffer RPE aliquots
• 1 x DNase I aliquot
• 1 x RNase-free water aliquot
• 5 x Qiagen RNeasy spin columns
• 10 x 1.5ml cent tubes
• 15 x 0.2ml cent tubes
• RNase-free, sterile tips & pipettors
• A temperature-controlled centrifuge
• Microcentrifuge tube pestle

Work on ice at all times, except where stated.
Work quickly and asceptically.

1. Add ~50mg of tissue (you may have to vary this amount for optimal extraction, depending on how much RNA is in the tissue) to 0.2ml of TRI and homogenise before adding another 0.8ml TRI
• Homogenise each sample in TRI before weighing out another
• Vortex and then aliquot 0.5ml and freeze as a duplicate
• Add 0.5ml of TRI to other half and stand for 5 min

2. Add 0.2ml Chloroform
• Vortex for 15 sec
• Stand for 15 min (room temperature)

3. Centrifuge TRI extraction for 15 min @ 12,000 @ 4C
• Pipette 200µl RNA phase (top/clear) into a clean eppendorf and add 200µl 70% EtOH.
• Avoid white cloud above white layer as this is genomic DNA.

4. Pipette the whole 400µl sample onto a Qiagen Rneasy mini spin column placed in a 2ml collection tube straight away! Close lids gently, and centrifuge for 15s at 12,000rpm. Discard the flow-through.

5. Add 350μl Buffer RW1 to each RNeasy spin column. Close the lid gently, and centrifuge for 15s at 12,000 rpm to wash the spin column membrane. Discard the flow-through.

6. Add 55μl DNase I stock solution (see above) to Buffer RDD aliquot. Mix by gently inverting the tube, and centrifuge briefly to collect residual liquid from the sides of the tube.

7. Add 80µl of the DNase I incubation mix directly to each RNeasy spin column membrane, and incubate on the benchtop (20–30°C) for 15 min.

8. Add 350μl Buffer RW1 to the RNeasy spin column. Close the lid gently, and centrifuge for 15s at 12,000 rpm. Discard the flow-through.

9. Add 500 μl Buffer RPE to the RNeasy spin column. Close the lid gently, and centrifuge for 1 min at 12,000 rpm to wash the spin column membrane. Discard the flow-through.

10. Place the RNeasy spin column in a new 1.5 ml collection tube (supplied). Add 30–50μl RNase-free water directly to the spin column membrane. Close the lid gently, and centrifuge for 1 min at 12,000 rpm to elute the RNA.

11. Aliquot into 3 x 10µl volumes in 0.2ml centrifuge tubes and freeze immediately at -80°C.

I will post about the success, or otherwise, of this method as soon as our sodding Bioanalyzer is fixed. Grrrrrrrr . . . .


  1. Hi was it a success..please let me know ..I want to use this method

  2. why would u want to extract RNA from tissue?



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