Erickson:RNA

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'''Procedure'''
'''Procedure'''
-
* Collect fleas (~25 depending on amount of RNA required) in a 3 ml tube.  Add 1 ml Trizol reagent and homogenize with tissue homogenizer (from Wilson lab).
+
    Collect fleas (~25 depending on amount of RNA required) in a 3 ml tube.  Add 1 ml Trizol reagent and homogenize (you can use the homogenizor).
-
* Incubate 5 min at room temperature.
+
    Incubate 5 min at room temperature.
-
* Centrifuge at 10,000 rcf at 4ºC for 10 minutes to pellet insoluble debris.
+
    Centrifuge at 12,000 rcf (11,400 rpm) at 4ºC for 10 minutes to pellet insoluble debris.
-
* Transfer supernatant to a fresh microfuge tube.  Take care not to take the pellet or fat layer as phenol may be trapped in micelles and lead to contamination.
+
    Transfer supernatant to a fresh microfuge tube.  Take care not to take the pellet or fat layer as phenol may be trapped in micelles and lead to contamination.
-
* Add 200μl chloroform (no isoamyl alcohol) to each tube.
+
    Add 200μl chloroform (no isoamyl alcohol) to each tube.
-
* Shake vigorously by hand (vortexing may lead to DNA contamination).
+
    Shake vigorously by hand (vortexing may lead to DNA contamination).
-
* Incubate at room temp for 3 min.
+
    Incubate at room temp for 3 min.
-
* Centrifuge at 10,000 rcf at 4ºC for 15 min.
+
    Centrifuge at 10,000 rcf (10,400 rpm) at 4ºC for 15 min.
-
* Transfer upper aqueous phase (~0.6ml) to new RNAse-free microfuge tube.
+
    Transfer upper aqueous phase (~0.6ml) to new RNAse-free microfuge tube.
-
* Add 0.5 ml isopropanol.
+
    Add 0.5 ml isopropanol and 1 uL glycoblue
-
* Incubate at room temp. for 10 min.
+
    Incubate at room temp. for 10 min.
-
* Centrifuge at 12,000 rcf at 4ºC for 10 min.
+
    Centrifuge at 12,000 rcf at 4ºC for 10 min.
-
* Remove supernatant and wash pellet with 1 ml 75% ethanol.
+
    Remove supernatant and wash pellet with 1 ml 75% ethanol.
-
* Centrifuge at 7,500 rcf for 5 min. at 4ºC
+
    Centrifuge at 7,500 rcf for 5 min. at 4ºC
-
* Remove supernatant.
+
    Remove supernatant.
-
* Centrifuge briefly (short on mini) and carefully remove last of supernatant with a pipette.
+
    Centrifuge briefly (short on mini) and carefully remove last of supernatant with a pipette.
-
* Air dry for 10 min.
+
    Air dry for 10 min (do not dry under vacuum or heat)
-
* Resuspend pellet in 80μl Nuclease free water.  Optional:  Add 20 μl DNase solution and incubate 10 minutes.
+
    Resuspend pellet in 80μl Nuclease free water.  Optional:  Add 20 μl DNase solution and incubate 10 minutes.
 +
    Degrade DNA with Turbo DNA-free kit made by Ambion (instructions can be found on line).

Revision as of 14:06, 27 April 2009


Isolation of Total RNA from Fleas

  • IMPORTANT!!

You must avoid contamination of RNAse. 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

    Collect fleas (~25 depending on amount of RNA required) in a 3 ml tube.  Add 1 ml Trizol reagent and homogenize (you can use the homogenizor).
    Incubate 5 min at room temperature.
    Centrifuge at 12,000 rcf (11,400 rpm) at 4ºC for 10 minutes to pellet insoluble debris.
    Transfer supernatant to a fresh microfuge tube.  Take care not to take the pellet or fat layer as phenol may be trapped in micelles and lead to contamination.
    Add 200μl chloroform (no isoamyl alcohol) to each tube.
    Shake vigorously by hand (vortexing may lead to DNA contamination).
    Incubate at room temp for 3 min.
    Centrifuge at 10,000 rcf (10,400 rpm) at 4ºC for 15 min.
    Transfer upper aqueous phase (~0.6ml) to new RNAse-free microfuge tube.
    Add 0.5 ml isopropanol and 1 uL glycoblue
    Incubate at room temp. for 10 min.
    Centrifuge at 12,000 rcf at 4ºC for 10 min.
    Remove supernatant and wash pellet with 1 ml 75% ethanol.  
    Centrifuge at 7,500 rcf for 5 min. at 4ºC
    Remove supernatant.
    Centrifuge briefly (short on mini) and carefully remove last of supernatant with a pipette.
    Air dry for 10 min (do not dry under vacuum or heat)
    Resuspend pellet in 80μl Nuclease free water.  Optional:  Add 20 μl DNase solution and incubate 10 minutes.
    Degrade DNA with Turbo DNA-free kit made by Ambion (instructions can be found on line).


RNeasy Cleanup
RNA may be purified by using either an RNeasy mini column, or a RNeasy MinElute column (depending on the amount of RNA you have and the concentration you desire in the end.

      1.   Binding capacity of each RNeasy mini column is 100 μg RNA.  Adjust total volume to 100 μl if 
     required (add 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 8000 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.
     Quantify a 1/100 dilution of RNA using nanodrop spectrophotometer.
     Run RNA gel (2-4 μl RNA, 1 μl loading dye, 1% agarose
     18.  Store at -80ºC

Revision by Kody Johnson

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