Protocol for amino allyl labeling

**Khan Lab (February, 2005)**

**[RT Reaction:]{.underline}**

After TRIzole purification and RNeasy, RNA is re-suspend in H~2~O. Take
1 μl for RNA concentration measurement, and make sure the total amount
of RNA for labeling is \> 15 μg, usually 20 μg. For amplified RNA, use 2
μg amplified RNA per labeling per slide.

Concentrate RNA to 23.2μl with Microcon-30.

**Total RNA** **Amplified RNA**

RNA 23.2μl 22.2 μl

Anchored primer (1μg/μl) 1 μl -

( or Random Hexamer for amplified RNA, 3μg/μl) - 2 μl

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Total volume 24.2μl 24.2μl

70^o^C for 5 minutes and cool to 42 ^o^C

RNAse Inhibitor RNAsin 1 μl

5X First Strand Buffer 8 μl

0.1 M DTT 4 μl

50X aa-dNTP mix 0.8 μl

SSII Reverse Transcriptase 2 μl

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Total volume 40 μl

42 ^o^C for 30 minutes

Additional 1 μl SSII RT enzyme

42 ^o^C for 30 minutes and cool to R/T

**[Labeling Reaction:]{.underline}**

1\. To each labeling reaction, add 5 μl of 500mM EDTA (pH8.0), mix well
before adding NaOH

2\. Add 10 μl 1 M NaOH, mix well

3\. 65 ^o^C for 20 minutes to hydrolyze RNA, then cool to R/T

4.  Add 10 μl 1 M HCl to neutralize NaOH

5.  Probe purification with Qiagen PCR spin column protocol.

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a.  Add 350 μl PB buffer to RT reaction and transfer to QIAquick column.

b.  Spin at maximum speed for 1 minute.

c.  Wash with 750 μl phosphate wash buffer (5 mM KPO4, pH 8.0 + 80% EtOH
    -- use this home-made buffer to avoid Tris).

d.  Spin at maximum speed for 1 minute.

e.  Repeat wash steps c and d.

f.  Empty collection tube and spin at maximum speed for 1 minute.

g.  Transfer column to a fresh tube, Elute with 30 μl elution buffer (4
    mM KPO4, pH 8.5) with 1 minute incubation followed by spin at
    maximum speed for 1 minute.

h.  Repeat elution step g.

i.  Speedvac dry (\~40 minutes)

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6.  Cy Dye coupling

    a.  Re-suspend the cDNA in 4.5 μl 0.1 M carbonate buffer, pH 9.0
        (freshly made, every two weeks, store at room temperature).

    b.  Add 4.5μl NHS-Cy dyes (re-suspended in DMSO. AVOID ANY
        MOISTURE!!)

    c.  Incubate the reactions in dark for 1 hour in room temperature.

**[Purification]{.underline}**

1.  Add 35 μl 100 mM NaOAc pH5.2 to the reaction.

2.  Add 250 μl PB buffer and follow QIAquick PCR purification protocol.

3.  Elute 2X in 30μl H2O.

> At this step, 1 μl of sample can be taken for dye incorporation
> analysis using NanoDrop Cy3/Cy5 program.

4.  Mix the probes of Cy3 and Cy5 in equal amount. Speedvac dry (\~40
    minutes).

**[Pre-hybridization:]{.underline}**

1.  Prepare pre-hybridization buffer (5xSSC, 0.1% SDS and 1% bovine
    serum albumin, BSA Sigma Cat# A-9418)

2.  Place slides to be analyzed into a staining dish, fill with
    pre-hybridization buffer, and incubate at 42 ^o^C for 45 minutes

3.  Wash the slides in a Wheaton slide rack at room temperature with
    DIH~2~O with agitation for 2 min.

4.  Dip the slides in room temperature isopropanol and dry by
    centrifugation at 700 rpm for 5 min. Inspect slides to make sure
    there is no protein marks on slides. If there is, repeat step 3 and
    4.

Slides should be used immediately following pre-hybridization, at least
within one hour.

**[Hybridization:]{.underline}**

1.  Re-disolve the combined probes in 27 μl 1X hybridization solution
    (240μl H2O, 250μl 20XSSC, 10μl 10%SDS, 500μl Formamide = 1ml) and
    combine with COT1-DNA and Poly(A)-DNA to block nonspecific
    hybridization

Cy5/Cy3 labeled probes 27 μl

Poly dA (8mg/ml) 1.3 μl

Yeast tRNA (4mg/ml) 1.3 μl

[CoT-1 DNA (10mg/ml, concentrated) 1.3
μl]{.underline}\_\_\_\_\_\_\_\_\_\_\_\_\_

Total 30.9 μl

> 2\. Denature at 95 ^o^C for 2 minutes
>
> 3\. Apply 14μl 5X SSC onto each modified hybridization chamber to
> prevent drying.
>
> 4\. Apply probes to the cover slide that is big enough for the printed
> area (e.g. 60mm cover slip for 22K array). Then put the pre-hybridized
> microarray slide on the top of the cover slip, invert, and hybridize
> in a humidified chamber at 42 ^o^C for 16-24 hours.

**[Wash:]{.underline}**

Place slide into a staining dish with Wash #1 at R/T until cover slip
falls off.

Wash #1: at R/T in 0.2% SDS + 1 X SSC until cover slip falls off

Wash #1: 4 minutes at 42 ^o^C in 0.2% SDS + 1 X SSC

Wash #2: 4 minutes at R/T in 0.2% SDS + 0.1 X SSC

Wash #3: 4 minutes at R/T in 0.06 X SSC

Spin immediately (700 rpm for 5 minutes at R/T)

**Appendix:**

Master mix for labeling reaction (4):

5X first strand buffer 8 μl X 4.5 = 36 μl

0.1 M DTT 4 μl X 4.5 = 18 μl

50X aa-dNTP mix 0.8 μl X 4.5 = 3.6 μl

RNAse inhibitor RNAsin 1 μl X 4.5 = 4.5 μl

Superscript II RTase 2 μl X 4.5 = 9 μl

Total: 15.8 μl/reaction

Calculation for each 1000ml washing buffer:

0.2% SDS: 20 ml 10%SDS in 1000 ml.

1X SSC: 50 ml 20XSSC in 1000 ml.

0.1XSSC: 5 ml 20XSSC in 1000 ml.

0.06XSSC: 3 ml 20XSSC in 1000 ml.

Solutions:

**[100 mM aa-dUTP]{.underline}** (Sigma, cat \# A0410)

Dissolve 10 mg in 191 ul 0.1 M KPO4 buffer, pH 7.5. Quantitate this
stock solution by diluting an aliquot 1:5000 in the same buffer and
measuring A289. Stock concentration in mM = OD~289nm~ x 704.

# **Make 10 ml 0.1 M KPO~4~ buffer, pH7.5**

1\. Prepare 2 solutions: 1 M KH2PO4 and 1M K2HPO4.

2\. Mix:

> 0.802 ml 1M K2HPO4
>
> 0.198 ml 1 M KH2PO4

3\. Bring to 10 ml with H~2~O. The pH of this KPO4 buffer should be at
7.5.

# **50X aa-dNTP mix**

Nucleotide/dye ratio with the ratio of aa-dUTP to dTTP. We use a 2:3
ratio. Prep are a 50X stock:

  ----------------------- ----------------------- -----------------------
  Start Concentration     Volume                  Final Concentration

  100 mM dATP             25 µl                   25 mM dATP

  100 mM dCTP             25 µl                   25 mM dCTP

  100 mM dGTP             25 µl                   25 mM dGTP

  100 mM dTTP             15 µl                   15 mM dTTP

  100 mM dUTP             10 µl                   10 mM aa-dUTP
  ----------------------- ----------------------- -----------------------

# 

# **Phosphate wash buffer**

1\. Prepare 2 solutions: 1 M KH2PO4 and 1M K2HPO4.

2\. Make 1 M KPO4 (the pH should be 8.5-8.7) mix:

> 9.5 ml 1M K2HPO4
>
> 0.5 ml 1 M KH2PO4

3\. Make 100 ml phosphate washing buffer, Mix in the following order:

> 15.25 ml H2O
>
> 84.25 ml 95% EtOH
>
> 0.5 ml 1 M KPO4, pH 8.5
>
> (This solution will be slightly cloudy)

# **Phosphate elution buffer**

Dilute 1 M KPO4 pH 8.5 to 4 mM for Phosphate elution buffer:

1 M KPO4 pH 8.5 0.4 ml

H2O 99.6ml

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Total 100ml

# **Carbonate buffer 0.1 M Na2CO3, pH 9.0:**

Na2CO3 0.27g

H2O 20ml

Adjust pH to 9.0 with 6 N HCl

Total with H2O 25 ml

# 

# **NHS-Cy dyes**

NHS-Cy3: AmershamPharmacia Cat# PA23001

NHS-Cy5: AmershamPharmacia Cat#PA25001

1.  Resuspend one tube in 73 ul DMSO.

2.  Use immediately, or aliquot 4.5 ul into 0.5 ml tubes and store at
    -80°C. Avoid moistures!!!