Protocol for Amino-allyl Reverse Transcription and NHS-Cy Dye Labeling

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MATERIALS

Equipment

- Single channel pipette: 0.5-10 µl, 10-100 µl, 200-1000 µl, Eppendorf

- Vacuum manifold, Millipore

- Speedvac

- Adjustable waterbaths (37° C, 42° C, 55° C, 100° C)

Disposables

- 96-well Multiscreen-FB filter plate, Millipore, MAFB NOB 10

- Catch plate, VWR, 622409-108

- Pipette tips: 1-10 µl, 2-20 µl, 20-200 µl, 200-1000 µl, ART

- Tubes: 1.5ml

Reagents

- Anchored dT25, dT₂₃VN, 8 µM stock GibcoBRL

- Random 9-mers, NNNNNNNNN, 1 µg/µl stock Gibco-BRL

- GFP poly A⁺ RNA, prepared from plasmid pSP64-GFP

- SuperScript II, GibcoBRL, 200 U/µl, 18064-022 (includes 5X First Strand Buffer and DTT)

- Nucleotides, Pharmacia 27-2035-02, 100mM each*

- 5-(3-Aminoallyl)-2'-deoxyurindine 5'-triphosphate sodium salt (AA-dUTP), Sigma A-0410

- RNase Inhibitor, Boehringer Mannheim 799 017, 40U/ml

- Buffers, Sigma, 2.5M NaOH, 2M MOPS, 1M Tris-HCl pH 7.5 8.5

- 4M Hydroxylamine, Sigma H-2391

- 0.1M NaBicarbonate pH 9.0

- Monofunctional NHS-ester Cy3/Cy5, APBiotech PA23001/PA25001; resuspend each tube in 72µl H₂O, aliquot 4.5µl into 16 tubes, dry in speedvac

- Millipore Binding Buffer (5.3M Gua-HCl in 150mM KAc, pH 4.8)

- Millipore Wash Solution (80% Ethanol)

- Slide pretreatment buffer, 5XSSC/0.2%SDS

- Hybridization Buffer (50% Formamide, 5X SSC, 5X Denhardt�s, 0.1%SDS, 100µg/ml ssDNA (Sigma), 100µg/ml COT-I DNA (BRL), 100µg/ml polyA₇₂)

- Wash buffers, 1XSSC/0.2%SDS, 0.1XSSC/0.2%SDS, 0.1XSSC

* Nucleotide mix (20X): 10mM GTP, 10mM ATP, 10mM CTP, 4mM TTP, 4mM AA-dUTP

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PROTOCOL

Reverse Transcription

- Mix together:

2 µg poly A⁺ RNA (or 50mg total RNA)

2 µl oligo dT₂₃VN (8 mM)

2 µl random 9-mers (1mg/ml)

2.5 ng GFP poly A⁺ RNA

10.5 µl total volume

- Incubate at 70° C for 10 minutes, chill on ice 30 seconds, spin

- Add the following:

4 µl 5X First Strand Buffer

2 µl DTT (0.1M)

1 µl Nucleotide Mix (10mM G/A/C, 4mM T, 4mM AA-dUTP)

1 µl H₂O

0.5 µl RNase Inhibitor

- Mix contents of tube gently and incubate at RT for 10 minutes

- Add 1 µl SuperScriptII

20 µl total reaction volume

- Mix contents of tube gently and incubate at 42°C for 2 hours

RNA hydrolysis and purification of cDNA

- Add 2 µl 2.5 M NaOH

- Incubate at 37°C for 10 minutes

- Add 10 µl 2 M MOPS

- Add 200 µl binding buffer to probe and mix

- Dispense into glass fiber filter plate

- Place filter plate on top of vacu-system and apply vacuum.

- Wash 6x with 80% fresh Ethanol

- Place filter plate on top of catch plate along with centrifuge alignment frame

- Do one dry spin to remove residual ethanol (3500 rpm, 5�)

- Add 50 µl H₂O. Incubate 1 minute at RT

- Place filter plate on top of a clean catch plate along with a centrifuge alignment frame and spin (3000 rpm, 5�)

- Repeat with another 50 µl H₂O

- Scan probe at OD 260nm, 550nm and 650nm.

- Dry in Speedvac until volume is less than 5µl (~45 minutes @ 50�C)

Coupling to NHS-ester Cy dyes

- Adjust volume of sample to 4.5µl

- Resuspend mono-functional NHS-ester Cy3 or Cy5 dye aliquot in 4.5µl of 0.1M NaBicarbonate Buffer pH 9.0

- Mix dye and cDNA

- Incubate 1 hr at RT (Note: there is no advantage to incubating longer)

Quenching the Reaction and Removal of uncoupled Cy dyes

- Add 4.5µl of 4M Hydroxylamine

- Add 16.5µl of H₂O to bring the volume to 30µl

- Add 150µl of Millipore binding buffer, purify as above in Millipore 96 well plate, elute twice in 50µl aliquots of H₂O

- Scan probe at OD 260nm, 550nm and 650nm and calculate probe yield, incorporated Cy-dye and specific activity

- Purify probe over G50 column (prespin column (3000 rpm 1�), transfer to new tube, apply probe, spin (3000rpm 2�), collect flow-through).

- Dry purified probe down in speedvac (~1.5 hours at 50 °C)

Hybridization

- Slide pretreatment: submerge mirrored slides for 40 minutes in 5XSSC/0.2%SDS at 55 °C (waterbath), rinse slide by two quick dips in ddH₂O, air dry. Rinse coverslip in ddH₂O and ethanol, air dry

- Resuspend dried down probe in Hybridization Buffer (25µl/reaction)

- Boil probe for 3 minutes, ice 30 seconds, spin briefly

- Apply probe to slide and cover with coverslip

- Incubate overnight (14-16 hrs) at 42 °C in humid chamber

- Preheat wash buffers to 55 °C

- Remove coverslip by immersing slide in 1XSSC/0.2%SDS

- Wash in 1XSSC/0.2% SDS for 10 minutes at RT

- Wash twice in 0.1XSSC/0.2%SDS at RT for 10 minutes

- Wash twice in 0.1XSSC at RT for 1 minute

- Rinse by two quick dips in ddH₂O, dry with compressed air

- Scan (PMT 700, green (532nm), filter 1; PMT700, red (633nm), filter 2; width 10mm, length 60mm).