P-EMBL-240 - P-EMBL-240
1) RNA amplification by in vitro transcription
(1) T7-dT primer, HPLC purified (Biospring, sequence: 5'-GGCCAGTGAATTGTAATACGACTCACTATAGGGAGGCGGTTTTTTTTTTTTTTTTTTTTTTTT-3'),
(2) 5 x first strand buffer (SuperScript II RT, Invitrogen, Cat. No. 18064-014),
(3) 0.1 M DTT (SuperScript II RT, Invitrogen, Cat. No. 18064-014),
(4) 10 mM dNTP-mix (single dNTPs from peqlab, 100 mM),
(5) SuperScript II RT 200U/µl (Invitrogen, 10000U, Cat. No. 18064-014),
(6) 5 x second strand buffer (Invitrogen, Cat. No. 10812-014),
(7) E. coli DNA Ligase 10U/µl (Invitrogen, 100U, Cat. No. 18052-019),
(8) E. coli DNA Polymerase I 10U/µl (Invitrogen, 1000U, Cat. No. 18010-025),
(9) E. coli RNase H 2U/µl (Invitrogen, 30U, Cat. No. 18021-014),
(10) DEPC-treated water (Ambion, Cat. No. 9906),
(11) Ethanol absolute (Merck, Cat. No. 1.00983.2500),
(12) EDTA pH 8.0, 0.5 M (Ambion, Cat. No. 9260G),
(13) PCI (Amresco, Cat. No. 0883),
(14) Glycogen 20 mg/ml (Roche, Cat. No. 901 393),
(15) Ammoniumacetate (Merck, Cat. No. 1116), 7.5 M,
(16) MEGAscript T7 High Yield Transcription Kit (Ambion, Cat. No. 1334),
(17) RNeasy Mini Kit (Qiagen, Cat. No 74106).
1.2) Protocol First Strand Synthesis
(1) Mix 10 µg of total RNA with 100 pmol of T7-dT(24) primer and 1 µl spike-in control RNA mix (4 ng RNA each) and adjust the volume with DEPC-treated water to a final volume of 18 µl.
(2) Mix and incubate at 70°C for 10 min, then chill on ice and spin down.
(3) Add 6 µl of 5 x first strand buffer, 3 µl of 0.1 M DTT, 1.5 µl of 10 mM dNTP-mix and 1.5 µl of SuperScript II RT.
(4) Incubate at 37°C for 1 hour, then spin contents and chill on ice.
1.3) Protocol Second Strand Synthesis
(1) Add 15 µl of 5 x second strand buffer, 1.5 µl of 10 mM dNTP-mix, 0.5 µl of E. coli DNA Ligase, 2 µl of E. coli DNA Polymerase I , 2.5 µl of E. coli RNase H (2 U/µl) and 33.5 µl of DEPC-treated water to the 18 µl of first strand mix.
(2) Incubate at 16°C for 2 hours.
(3) Add 75 µl DEPC-treated water and 10 µl 0.5 M EDTA and chill on ice.
(4) Purification by phenol/chloroform extraction:
Add 160 µl PCI, vortex for 1 min and spin at 12000 rpm for 5 min.
Transfer the upper phase to a fresh tube and add 1 µl glycogen, 80 µl 7.5 M ammonium acetate and 400 µl ice-cold ethanol. Vortex and centrifuge at 12000 rpm for 20 min.
Remove the supernatant and wash the pellet twice with 70% ethanol.
Air-dry the pellet and resuspend in 6 µl DEPC-treated water.
1.4) Protocol for In Vitro Transcription (IVT)
(1) Add to 6 µl cDNA sample 1.5 µl 10x reaction buffer, 1.5 µl of each ribonucleotide (ATP, CTP, GTP, UTP) and 1.5 µl T7 polymerase enzyme mix.
(2) Incubate at 37°C for 4 hours.
(3) Add 0.5 µl of DNase I and incubate at 37°C for 15 min. Chill on ice.
(4) Purification with Qiagen RNeasy Mini Kit according to the manufacturer's guidelines.
(5) Check aRNA concentration by OD. Quality control is performed using Agilent Bioanalyzer.
2) Aminoallyl Labeling
(1) Random primers, 3 µg/µl (Invitrogen, Cat. No. 48190-011),
(2) DEPC-treated water (Ambion, Cat. No. 9906),
(3) Ethanol absolute (Merck, Cat. No. 1.00983.2500),
(4) EDTA pH 8.0, 0.5 M (Ambion, Cat. No. 9260G),
(5) Atlas Glass Fluorescent labelling Kit (Clontech, Cat. No. K1037-1),
(6) Cy3 and Cy5 mono-reactive dye packs (Amersham Pharmacia, Cat. No. PA23001 and 25001).
2.2) Cy-dye labeling protocol
2.2.1) cDNA synthesis with aminoallyl incorporation and purification
(1) Mix 5 µg of aRNA from IVT, 1 µl of random primers and DEPC-water to a final volume of 25 µl.
(2) Denature at 70°C for 10 minutes and chill on ice.
(3) In parallel pipette the following kit components into another tube: 7.5 µl deionised water, 10 µl 5x cDNA synthesis buffer, 5 µl dNTP mix and 2.5 µl MMLV reverse transcriptase.
(4) Add the denatured RNA mix to the transcriptase mix and incubate 40 minutes at 45°C, then stop the reaction at 70°C for 5 min and let the tube cool down at room temperature.
(5) Add 0.5 µl RNase H and incubate at 37°C for 15 min.
(6) For purification follow user manual.
2.2.2) Fluorescent dye coupling and probe purification
-Follow the manufacturer's guidelines.
Amount of nucleic acid labeled, Amplification, Label used