[Protocol for mRNA amplification and target preparation]{.underline}

Wang et. al., *Nature Biotechnology*, April, 2000

## Isolate Total RNA using Qiagen midi kit (Cat#75142) (see manufacturer\'s protocol) or by Trizol (Gibco BRL Cat# 15596-026) extraction (see manufacturer\'s protocol). Resuspend total RNA in DEPC water with addition of 1ul of RNasin before store at -80.

**First strand cDNA synthesis:**

In PCR reaction tube, mix 0.5-3ug total RNA in 9ul DEPC H2O with 1ul
(0.25ug/ul) oligo dT(15)-T7 primer (5' AAA CGA CGG CCA GTG AAT TGT AAT
ACG ACT CAC TAT AGG CGC T(15) 3'), 1ul of RNasin (Promega Cat# N2111)
and heat to 70C for 3min, cool on ice. Then add the following reagents:
(you can make a mastermix for multiple samples)

4ul 5 X First strand buffer

> 1ul (0.25ug/ul) TS (template switch) oligo primer (5' AAG CAG TGG TAA
> CAA CGC AGA GTA CGC GGG 3')

2ul 0.1M DTT

2ul 10mM dNTP (Pharmacia Cat# 27-2035-02)

1ul Superscript II (Gibco BRL Cat# 18064-071)

42C for 90min in thermal cycler.

(Note: buffer and 0.1M DTT come with SS II)

**Second strand synthesis:**

Add 106 ul of DEPC H2O to the cDNA reaction tube

15ul Advantage PCR buffer

3ul 10 mM dNTP mix

1ul of RNase H (2U/ul Gibco BRL Cat# 18021-071)

3ul Advantage Polymerase (Clontech Cat# 8417-1)

> 37C for 5min to digest mRNA, 94C for 2min to denature, 65C for 1min
> for specific priming and 75C for 30min for extension.

Stop reaction with 7.5ul 1M NaOH solution containing 2mM EDTA and
incubate at 65C for 10min to inactivate enzyme.

**Double strand cDNA cleanup:**

Add 1ul Linear Acrylamide (0.1ug/ul, Ambion Cat# 9520) to sample. Add
150ul Phenol: Chloroform: Isoamyl alcohol (25:24:1) (Boehringer Mannhem
Cat #101001) to ds cDNA tube and mix well by pipeting (be careful not to
spill or contaminate). Transfer the slurry solution to Phase lock gel
tube (5'-3' Inc. Cat# p1-257178) and spin at 14,000rpm for 5min at room
temperature. Transfer the aqueous phase to RNase/DNase-free tube, add
70ul of 7.5M ammonium acetate (Sigma Cat# A2706) and gently mix. Add 1ml
100% ethanol. Centrifuge at 14,000rpm for 20min at room temperature.
Wash pellet with 500ul 100% EtOH and spin pellet down at maximum speed
for 6min. Air dry or speedvac dry pellet.

**In Vitro Tanscription (Ambion; T7 Megascript Kit #1334)**

Add the following mixture to the dry pellet:

2ul of each 75mM NTP (A, G, C and UTP)

2ul reaction buffer

2ul enzyme mix (RNase inhibitor and T7 phage polymerase)

8ul DEPC H2O

37C for 6hr.

**aRNA purification using TRIzol reagent (GibcoBRL, #15596)**

Add 1ml of TRIzol solution to each in vitro transcription tube and mix
well. Add 200ul Chloroform per 1ml TRIzol solution. Mix by shaking
vigorously for 15 second and incubate at room temperature for 2-3 min.
Centrifuge at 12,000g for 15min at 4C. Transfer aqueous phase to a new
RNase free tube and add 500ul of isopropyl alcohol per 1ml TRIZOL
reagent. Incubate sample at RT for 10min and then centrifuge at 14,000
rpm for 15min. Wash pellet 2 times with 1ml 70% EtOH in DEPC-treated
water. Air dry pellet and quickly resuspend in 20ul DEPC-treated water.
(Note: over-dried RNA is difficult to dissolve into water.) Check RNA
concentration and quality by measuring OD260 and OD260/280. (Note: for
OD readings, dilute in DEPC H2O with 50 mM NaOH to linearize RNA.).

Note: When starting material is in nano gram scale, amplified RNA should
be isolated using half of the volume of TRizol reagent. Reagent used in
the following step should be reduced in half accordingly. To enhance the
precipitation, add 1ul Linear Acrylamide (0.1ug/ul, Ambion Cat# 9520) to
the transferred aqueous phase before centrifugation.

# Second round amplification

Mix aRNA(0.5-1ug) in 9ul DEPC H2O with 1ul (2ug/ul) random hexamer (i.e.
dN6) and heat to 70C for 3min, cool to room temperature. Then add the
following reagents:

4ul 5 X First strand buffer

1ul (0.5ug/ul) oligo dT-T7 primer

2ul 0.1M DTT

1ul RNAsin (Promega Cat# N2111)

2ul 10mM dNTP (Pharmacia Cat# 27-2035-02)

1ul Superscript II (SS II) (Gibco BRL Cat# 18064-071)

42C for 90min.

**From here, follow the procedure of first round amplification for ds
cDNA synthesis and cleanup.**

**Target labeling by reverse transcription:**

4ul First strand buffer

1ul dN6 primer (8ug/ul; Boehringer Mannheim Cat# 1034731 resuspended in
250ul DEPC H2O)

2ul 10X low T - dNTP (5mM A, C and GTP, 2mM dTTP)

2ul Cy-dUTP (1mM Cy3 or Cy5)

2ul 0.1 M DTT

1ul RNasin

3-6ug amplified mRNA in 7 ul DEPC H2O

Mix well and heat to 65C for 5min then cool down to 42C.

> Add 1.5ul SSII and Incubate for 90 min at 42C. Add 2.5ul 500mM EDTA
> and heat to 65C for 1min. Add 5ul 1M NaOH and incubate at 65C for
> 15min to hydrolyze RNA. Add 12.5ul 1M Tris immediately to neutralize
> the pH. Add 35ul of 1xTE.

**Target clean up:**

Prepare Bio-6 column and run target solution through it. Collect flow
through and add 200ul 1xTE. Concentrate target to \~20ul using a
microcon YM-30 column (Millipore Cat# 42410).

**Hybridization:**

Combine Cy3 and Cy5 labeled target (adjust the color to purple) and
complete dry the sample by speed vacuum . Add 37ul of the following
mixture to the dried target (for 20x40mm slide):

1ul 50x Denhardt's blocking solution (Sigma Cat# 2532)

1ul poly dA (8mg/ml Pharmacia Cat# 27-7988-01)

1ul yeast tRNA (4mg/ml Sigma Cat# R8759)

10ul Human Cot I DNA (1mg/ml Gibco BRL Cat# 15279-011)

3ul 20X SSC

1ul 10% SDS

20ul of DEPC H2O

Heat for 2 min at 99^o^C and Cool to RT. Apply target mixture to array
slide, add coverslip, place in humidified hyb chamber, and hybridize at
65^o^C over night.

# Washing:

1.  Wash with 2x SSC + 0.1% SDS to remove coverslip.

2.  Wash with 1x SSC for 1 min.

3.  Wash with 0.2x SSC for 1 min.

4.  Wash with 0.05x SSC for 10-20 seconds.

5.  Centrifuge slide at 80-100 g for 3 min. (Slide can be put in slide
    rack on microplate carriers or in 50ml concial tube and centrifuged
    in swinging-bucket rotor.)

**Scan Slide**