AutoGen Protocol - Animal Tissue RNA

I. Purpose: To extract Total RNA from animal tissue.

II. Chemical Principal: Modified AGPC (Acid Guanidine Phenol Chloroform) extraction method.

III. Protocol Parameters:

A.    Sample Volume: ~40mg (liver)
                             ~70mg (other types of tissue*)
                             * Some type of tissues are not suitable for RNA extraction.

B. Maximum Number of Samples: 24 samples per run.

C. Processing Time: 2.0 hours for 12 samples*
2.5 hours for 24 samples*
* This includes drying and resuspending RNA steps.

D. Yield*: ~150μg of total RNA / 30mg of mouse liver
           ~25μg of total RNA / 30mg of mouse heart
           *Measured by OD260 value
           *Actual yield may vary, depending on the condition and type of tissue sample.

E. Quality: Typical OD260/280 values in water are 1.85 - 1.95*.
             The RNA can be used directly in downstream processes such as RT-PCR,
             real time RT-PCR and Microarray analysis.
             * For most commonly used tissue samples.

IV. Running the Protocol :

                 A.    Load Reagents, Tips and Samples
                 B.     Select a Protocol
                 C.     Enter Number of Samples
                 D.     Start the Run

V. Example of AutoGen purified RNA:

                 A. Extracted total RNA:

Gel Electrophoresis:

Sample: Approx. 3 μg of total RNA
                was denatured at 65°C for 10
                min. and loaded onto the gel.

Gel: Formaldehyde denatured 1% agarose
         1 x MOPS, 50V x 1.5hr

B. Typical Yield and Purity⁺

+ Actual yield and purity may vary, depending on the condition of tissue sample.

VI. Extraction Process:

Process Site	Purpose	System Process
1. Manual	Homogenize Tissue	Add Reagent R0*, homogenize tissue and transfer to AutoGen 2ml tube.
2. Automated	Remove Protein and DNA Part-1	Add Reagent R1* and R2*, and mix.
3. Automated	Remove Protein and DNA Part-2	Add Reagent R3*, incubate, centrifuge to pellet protein & DNA, and transfer supernatant to new tube.
4. Automated	Precipitate RNA	Add Reagent R4*, mix, centrifuge to precipitate RNA, and discard.
5. Automated	Wash RNA	Add Reagent R5*, mix, centrifuge and discard supernatant. Repeat 3 times.
6. Automated	Dry RNA	Centrifuge tube to dry RNA sample.
7. Automated	Resuspend RNA	Add Reagent R6*.
* All reagents and plastics are sold as a complete extraction kit for Tissue RNA on the AutoGenprep 245T.

Version 1.0 - March/2004