Q. Guo, L. Miller, J. Villa

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Probe Preparation/Hybridization Using Total RNA

 

I.        Probe Making

  1. Make the following RT labeling reaction mix for each probe:

 

Component

Vol (µl)

5X first strand buffer

8

Oligo dT 20-mer   (2 µg/µl)

2

20X low-dT/(NTP) mix

2

Cy3 or Cy5 dUTP (1 mM)

4

0.1 M DTT

4

RNAsin

1

(50-100µg) total RNA

19 (combine with dH20 for 19 µl)

 

 

TOTAL

40 µl

 

NOTE: As a starting point, we recommend using 50 µg total RNA for Cy3 labelling and 100 µg total RNA for Cy5 labelling. For RNA preparation, we suggest Trizol-LS extraction followed by double precipitation (e.g. precipitate, wash, resuspend X2)

 

  1. Vortex reaction mix, quick spin.
    AVOID BUBBLES AND FOAMING AT ALL TIMES.

 

  1. Incubate at 65°C for 5 minutes, place at room temperature for 2.5 minutes, and transfer to 42°C.
  2. Add 2 µl of SSII enzyme.
    MIX ENZYME WELL INTO THE REACTION.
  3. Incubate 42°C for 25 minutes.
  4. Add 2 µl of SSII enzyme.
    MIX ENZYME WELL INTO THE REACTION.
  5. Incubate at 42°C for 35 minutes.
  6. Add 5 µl of 500 mM EDTA and vortex.
    STOP YOUR REACTION WITH EDTA BEFORE ADDING NaOH.
  7. Add 10 µl of 1M NaOH and vortex/spin.
  8. Incubate at 65oC for 60 minutes to hydrolyze residual RNA.
  9. Cool to room temperature.
  10. Add 25 µl of 1 M Tris-HCl (pH7.5).

 

II.      Probe Cleanup

 

  1. Turn on dry bath heatblock to 100C.
  2. Add 500 µl of TE (7.4) to Microcon column and spin 7-8 minutes at 13K to wash column.
  3. Add 400 µl TE to the sample tube and transfer sample to the Microcon column.
  4. Spin until about 20-40 µl left (13K, ~8 minutes).
  5. To combine probes, carefully invert one column of probe (Cy3) into a fresh tube (supplied with Microcon kit) and spin at 13K for 1 minute.
  6. Transfer recovered probe to the Microcon column containing the other probe (Cy5).
  7. Add 400 µl TE into the column and spin down to 20-30 µl.
  8. Wash the probe a final time (to remove unincorporated fluor) by adding 450 µl TE and reduce volume to 6-8 µl as measured carefully by pipetman.
  9. Invert column into a fresh tube and spin 1 minute at maximum speed to recover probe. (If recovered probe volume is <11 µl, increase volume up to 11 µl with TE.)
  10. For a final volume of 17.6 µl, add 1 µl (10 µg) COT-1 DNA, 1 µl (8-10 µg) polyA, 1 µl yeast tRNA (4 µg), 3.1 µl 20X SSC ( for 3.5X final),  and 0.5 µl 10% SDS (for 0.3% final).
  11. Heat for 2 minute at 100oC.
  12. Spin 10 minutes at 14000 xg to pellet any particulate matter and to facilitate cooling.
  13. Transfer probe (minus any pelleted particulate matter) to a clean microfuge tube taking care to minimze bubble formation.

 

III.     Hyb Setup

1.      Resuspend probe thoroughly via gentle pipeting being careful to minimize bubble formation. Add total volume of probe (~17.6 µl) to the center of a processed array and quickly (but carefully!) add cover slip (22mm x 22mm) taking care to prevent bubble accumulation beneath slip. Use forceps to lower cover slip over probe and gently drop onto probe such that the probe is spread evenly over the surface of the array. If bubbles are seen under the cover slip, don't worry. They usually move to the edge during the incubation.

  1. Place slide in hyb chamber, add 20 µl water to far end of slide (to maintain humidity), firmly attach margin clamps, and incubate overnight (10-16 hours) @ 65oC. note: It is very important to maintain humidity. Water volume may vary depending upon the type of hyb chamber used. For the hyb chambers manufactured by Telechem, we add ~40 µl total - that is, 20 µl to each end well.

 

IV.     Hyb Washes

  1. Carfully remove margin clamps of hyb chamber to prevent water from seeping in and contaminating the array.
  2. Remove slide from chamber, place in slide rack, and submerge in staining dish filled with 2X SSC and 0.1% SDS. Plunge gently until cover slip falls away from the slide. (Tilt slide at an angle as you plunge to ensure cover slip falls away from array without scratching it.)
  3. Wash for 1 minute in 1X SSC, plunging gently.
  4. Wash for 1 minute in 0.2X SSC, plunging gently.
  5. Wash for 10-20 seconds in 0.05X SSC, plunging gently.
  6. Quickly spin dry (in slide rack) in a centrifuge set at 50-100 xg for 3-4 minutes.
  7. Scan as soon as possible (scan Cy5 (red) channel first since it is prone to fading).

 

WASHES:

2XSSC+0.1%SDS

1XSSC

0.2XSSC

0.05XSSC

dH20:

179 ml

190 ml

198 ml

200   ml

20XSSC:

 20 ml

 10 ml

  2 ml

  0.5 ml

20%SDS:

  1 ml

  -

  -

   -

 

V.       Reagents

 

            

(1)

Oligo dT (20mer):

2.0 µg/µl*

GIBCO-BRL 

custom, cartridge-purified

(2)

Cy3-dUTP:

1 mM

Amersham

Cat # PA53022

(3)

Cy5-dUTP:

1 mM

Amersham

Cat # PA55022

(4)

SuperScript II:

200 U/µl

GIBCO-BRL

Cat # 18064-014

(5)

RNAsin

20-40 U/µl ,

Promega

Cat # N2515

(6)

Yeast total tRNA

4 µg/µl*

Sigma

Cat # R8759

(7)

human COT-1 DNA

10 µg/µl+

Boehringer Mannheim

Cat # 1-581-074

(8)

polyA 

8 µg/µl*

Pharmacia

Cat # 27-7988-01

(9)

100 mM dNTP set

20X, low-dT^

Pharmacia

Cat # 27-2035-01

(10)

Microcon

YM-30 columns

 

Amicon

Cat # 42410

 

Other reagents:  20X SSC, TE pH7.4, 10% SDS, 500 mM EDTA, 1M NaOH,

1M Tris-HCl pH7.5, sterile dH2O

 

* comes lyophilized, must be resuspended at specified concentration

+ comes as 1 µg/µl, must be precipitated and resuspended at specified concentration

^ comes as 100 mM, for 20X stock: 10 mM each of dA, dG, dC and 4 mM of dT in dH2O