ARK Genomics

Standard Operating Procedure

S.O.P# 

 

: IGF098.00  

Title 

 

:

Dye coupling reaction using amplified RNA incorporating aminoallylUTP

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1.0

BACKGROUND
The MessageAmp aRNA Kit is based on the RNA amplification protocol developed in the laboratory of Dr James Eberwine.  The procedure consists of reverse transcription with an oligo (dT) primer bearing a T7 promoter and in vitro transcription of the resulting DNA with T7 polymerase to generate hundreds to thousands of antisense RNA copies of each mRNA in a sample.  The antisense RNA is referred to as aRNA and the amplification method is referred to as the aRNA amplification procedure.  AminoallylUTP UTP can be incorporated into the aRNA during the in vitro transcription reaction.  This can then be used directly in a dye coupling reaction.

 

2.0

PURPOSE
This protocol outlines the method used to label small quantities of RNA for use in microarray analysis.

 

3.0

RELATED SOPs
RNA Amplification using the Ambion MessageAmp kit             SOP097.00

4.0 MATERIALS
 

Cy3 monoreactive dye pack                                        CS#006
Cy5 monoreactive dye pack                                        CS#007
Coupling Buffer from the Fairplay kit                           CS#003
DMSO from the Fairplay kit                                          CS#003
DyeEx 2.0 spin kit                                                        EQU#053

5.0

METHOD

  5.1 Set driblock at 37ºC.  If necessary, resuspend a fresh aliquot of Cy dye in 45 ml DMSO (supplied in the Stratagene Fairplay kit), and vortex gently to solubilise. Store dye in aliquots of 10ml (for two reactions) in amber tubes -20 °C in the dark. These Cy dyes are stable for several months.
  5.2 Place 1.2ug aaUTP aRNA in 20ul MilliQ water in an RNase free microfuge tube.
  5.3 Dry down in the SpeedVac centrifuge until the liquid has just been removed from the tube.  Do not over dry since this will make the RNA more difficult to resuspend.  During this time place the coupling buffer at 37C to dissolve any precipitate.
  5.4 Resuspend the RNA in 5ul coupling buffer from the Stratagene Fairplay kit.  Vortex and spin briefly to collect the contents at the bottom of the tube.
  5.5 To aid resuspension place the tubes at 37ºC for thirty minutes.  After this time vortex and centrifuge briefly to return the contents to the bottom of the tube.
  5.6

Add 5ul of Cy dye to the cDNA and mix by gently pipetting up and down, incubate for one hour at room temperature in the dark.  Proceed immediately to procedure 6.1

 

6.0 DYE COUPLED cDNA PURIFICATION
  6.1 Take one DyeEx 2.0 spin column per reaction and vortex to resuspend the resin.
  6.2 Label the tube, loosen the cap of the spin column a quarter turn, snap off the bottom closure and place into a 2ml collection tube.
  6.3 Spin the columns using the Eppendorf microcentrifuge 5415D at 3000rpm for three minute. Add 300ml MilliQ water to the resin and spin for another three minutes.  When returning the tubes to the centrifuge ensure that the column is placed in the same orientation in the centrifuge.
  6.4 Carefully transfer the spin column to an amber tube and slowly apply the labelling reaction to the gel bed. Pipette the labelling reaction directly onto the centre of the slanted gel-bed surface.  Do not allow the reaction mixture or the pipette tip to touch the sides of the columns.  Pipette the sample slowly so that the drips are absorbed by the resin.
  6.5 Return the tubes to the centrifuge; again ensuring the column is placed in the same orientation in the centrifuge as previously.  Spin the columns at 3000rpm for three minutes.
  6.6 Remove the spin column from the tube and retain in a rack until after the QC of the labels. The eluate contains the purified labelling reaction.  The volume of the eluate is approximately 10ul. Discard columns once QC of labels is complete.