RNA ReverseTranscriptase Labeling with Aminoallyl – dUTP
REAGENTS
FluoroLink Cy3 and Cy5 monofunctional dyes
(Amersham catalog # PA23001, and PA25001)
NHS-ester = 1[episil-carboxy-pentyl]1’ethyl 3, 3, 3’,3’
Bring up in 14 ul of High quality DMSO and use 1 ul
OR
Re-suspend one dye pack in 14 ul DMSO, dry 1 ul aliquots and store at –80C in foil (dark)
Reverse Transcriptase
SuperScript II RNase H- Reverse Transcriptase (Gibco BRL #18064-014)
aa_dUTP
5-(3-aminoallyl)-2’-deoxyuridine 5’-triphosphate (AA-dUTP) (Sigma A0410)
1 mg = 1.91 10-6 Mole bring up in 19.1 ul (19 *10-6 l) = 100mM. Store stock at –80C.
50X aa dUTP/dNTPs:
10 ul dATP (100 mM)
10 ul dGTP (100mM)
10 ul dCTP (100mM)
6 ul dTTP (100mM)
4 ul of aa dUTP (100 mM)
dTV (25-mer) primer 50 pmol/ul
5’ - TTT TTT TTT TTT TTT TTT TTT TTT V – 3’
V = A, C, or G
Sodium bicarbonate 0.33 M pH 9.2
carbonate buffer=
1.36 g sodium carbonate
7.35 g sodium bicarbonate
950 ml H2O,
adjust pH to 9.2 with 1M HCl or 1 M NaOH if necessary
H2O to 1 liter
OR
0.2 M anhydrous sodium carbonate ( 21.2 g in 1 liter)
0.1 M solution of sodium bicarbonate (16.8 g in 1 liter)
For pH 9.3 mix 7.5 ml of anhydrous with 42.5 ml of bicarb and dilute to a total of 200 ml.
PROTOCOL
I.Reverse Transcriptase Reaction
Oligo dTV primer (50pmol/ul) 2 ul
10 ug total RNA 13.5 ul
Total 15.5 ul
Before adding RNA: ethanol precipitate and bring up pellet in enough water than there will be 10ug in 13.5 ul. Spec on the Nanodrop.
If you have 10ug in smaller volume, add depc-H2O to 15.5ul. If have 10ug in larger volume, subtract corresponding amount of depc-H2O from reverse transcriptase reaction.
Incubate at 92°C for 1 min.
Chill on ice.
cDNA synthesis
|
Component |
ul |
For 10 rxns (by 10.5) |
|
5X first strand buffer (from SSII RT) |
6 |
63 |
|
50X aa dUTP/dNTPs |
0.6 |
6.3 |
|
DTT (0.1 M) |
3 |
31.5 |
|
SuperScript II ReverseTranscriptase |
1 |
10.5 |
|
DEPC water |
3.9 |
41 |
|
Total |
14.5 |
14.5 X 10.5=152 |
*Note, DTT is an anti-oxidant that keeps an enzyme at max. activity
*Note, most reagents are in the ligase/buffer box at –20 C. SSII RT is in the polymerase box.
Incubate at 42 C for 2 hours or longer (o/n is ok) – use minicycler
Heat to 94°C for 2min
Quick cool on ice
Add 1 ul of RNase mix (Ambion) – stored in nuclease box
Incubate at 37°C for 15 minutes
II. Hydrolysis
Add: 1N NaOH 10 ul
0 .5M EDTA 10 ul
Incubate at 65C for 15 min
Neutralize: 1M Tris pH 7.4 25 ul
III. Cleanup of cDNA
To continue with the amino-allyl dye coupling procedure all Tris must be removed from the reaction to prevent the monofunctional NHS-ester Cye dyes from coupling to free amine groups in solution.
Increase volume to 200ul by adding 140 ul 1mM EDTA
Ethanol precipitate (1/10 vol 3 M NaOAC, 2.5 vol EtOH) – store in –20C for 20 min, spin at 4C for 30 min
Bring up in 50 ul dH2O
Run through a G-50 column (Amersham #37-5330-01) to remove unincorporated nucleotides. You may prefer to make your own G-50 column. See below for instructions.
Spec on the Beckman. Should have 10-20% of starting yield.
Ethanol precipitate. Can leave overnight in –20C.
Remember THE POINT of the Cleanup: to remove all free aa-UTPs and all Tris buffer.
IV. Coupling
Bring dry cDNA up in 7 ul dH2O. If doing a loop design, bring cDNA up in 20ul. Each RNA sample should be labeled with both Cy3 and Cy5. Divide sample into Cy3 and Cy5 tubes.
Heat to 95º C for 2 min, quickly chill on wet ice
Add 3 ul sodium bicarbonate buffer (0.33 M pH 9.3).
Reconstitute Cy dye in 1 ul DMSO and add to cDNA.
Incubate at RT for 1 hour in dark (can wrap tube rack in foil or put rack in dark drawer).
V. Quenching and Cleanup
Add 25ul 1 M Tris-HCl pH 7.5.
Incubate at RT for 15 min in dark.
Add 10ug carrier DNA (10ug/ul sheared herring sperm DNA). To shear DNA, vortex and run through tiny syringe multple times.
Add 165 ul H2O to increase volume to 200ul
EtOH precipitate. Bring up in 50ul H2O.
Run through G50 or GeneClean columns two times or use PCR cleanup kit – to remove unincorporated Cy dyes.
Spec DNA on Beckman using Cy dye setting
Ethanol precipitate. You can leave overnight at –20C.
Pellet will be resuspended in hybridization buffer. See microarray hybridization protocol for this.
Making your own G-50 columns
Add 10g Sephadex (G-50) into approx. 250 ml DEPC water
Autoclave
Aliquot into 50 ml Falcon tubes
G50 should form sludge at bottom of tube. Make sure that there is enough water to saturate and form an H20 layer above G-50.
Add 1.5 ml of G50 to spin column (Bio-Rad #732-6008)
Spin at max speed (3000 rpm) for 2 min. Transfer column to new collection tube. Save eluate to use as blank for Beckman Spec.
Add 50 ul of sample Carefully to middle of column
Spin at max speed for 4 min
Blank eluate on Beckman Spec before measuring OD of sample.