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



 - 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)



 - 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



 - 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



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 ARNA

               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)


 - 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).

Department of Microbiology
School of Medicine
University of Washington
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