Erickson:RNA

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Protocol for extraction of Total RNA from Fleas

• IMPORTANT!!

You must avoid contamination of RNAse. RNA is delicate and easily degraded. Do not handle equipment or reagents with un-gloved hands and be sure to use RNAse free solutions and glass/plastic ware. It is a good practice to clean your equipment frequently with RNAse ZAP or equivalent.

Materials: • TRIzol reagent • Chloroform • Ethanol • Microcentrifuge tubes • Nuclease-free water • RNeasy mini kit

Procedure:

     1.  In a 1.5 ml microfuge tube, add 25 fleas (more or less depending on desired RNA yield), 1 ml TRIzol reagent and homogenize.

• Work quickly as RNA is susceptible to degradation during homogenization*

     2.  Incubate 5 min. at room temp.
     3.  Centrifuge at 12,000 rcf for 10 min. at 4ºC to pellet insoluble debris
     4.  Transfer supernatant to new microfuge tube.

• Take care not to take pellet or fat layer as phenol may be trapped in micelles and lead to contamination*

     5.  Add 200μl chloroform (no isoamyl alcohol) to each tube.
     6.  Shake vigorously by hand (vortexing may lead to DNA contamination).
     7.  Incubate at room temp for 3 min.
     8.  Centrifuge at 10,000 rcf at 4ºC for 15 min.
     9.  Transfer upper aqueous phase (~0.6ml) to new RNAse-free microfuge tube.

• Make sure to avoid RNAse contamination from this point on!*

     10.  Add 0.5 ml isopropanol.
     11.  Incubate at room temp. for 10 min.
     12.  Centrifuge at 12,000 rcf at 4ºC for 10 min.
     13.  Remove supernatant and wash pellet with 1 ml 75% ethanol.
     14.  Centrifuge at 7,500 rcf for 5 min. at 4ºC
     15.  Remove supernatant.
     16.  Centrifuge briefly (short on mini) and carefully remove last of supernatant with a                  
            micropipette.
     17.  Air dry for 10 min.
     18.  Resuspend pellet: add 20μl DNAse 
                            add 80μl Nuclease free water 

• optional*

     19.  Quantify a 1/100 dilution of RNA on spectrophotometer.

RNeasy Cleanup

     1.   Binding capacity of each RNeasy mini column is 100 μg RNA.  Bring RNA to concentration of no more than 1 μg/μl for total volume of 100 μl (added RNAse-free water)
     2.   Add 350 μl Buffer RLT and mix (be sure β-mercaptoethanol is added to Buffer RLT before use.)
     3.   Add 250 μl ethanol and mix by pipetting.
     4.   Apply sample to RNeasy column in 2 ml collection tube.
     5.   Centrifuge at 8,000 rcf for 15 s.
     6.   Transfer column to new collection tube and discard flow through.
     7.   Add 500 μl Buffer RPE to column (be sure ethanol is added to Buffer RPE before use.)
     8.   Centrifuge at 8.00 rcf for 15 s.
     9.   Discard flow through and reuse collection tube.
     10.  Add 500 μl Buffer RPE to column.
     11.  Centrifuge for 2 min at 8,000 rcf.
     12.  Transfer column to new 2 ml collection tube.
     13.  Centrifuge at full speed for 2 min (to dry column)
     14.  Transfer column to new 1.5 ml collection tube.
     15.  Add 50 μl RNAse free water to column bed (place in center!)
     16.  Incubate 2 min at room temp.
     17.  Centrifuge full speed for 1 min.

• optional*

     ^Quantify a 1/100 dilution of RNA on spectrophotometer.
     ^Run RNA gel (2-4 μl RNA, 1 μl loading dye, 1% agarose, 0.5x TBE mini-gel, 0.5 μl EtBr, run in 0.5 TBE) ~60 min. @ 100 V

• Note: 1X TAE may be substituted for 0.5X TBE

     18.  Store at -80ºC

Revision by Kody Johnson