P-MEXP-579 - P-MEXP-579

Type
labeling
Description
VIB-MAF Cy5 Labeling v1


1 RNA amplification by in vitro transcription


This protocol has been published in L. G. Puskás, Á. Zvara, L. Hackler Jr., and P. Van Hummelen (2002). RNA amplification results in reproducible microarray data with slight ratio bias. Biotechniques 32(6): 1330-1341 (2002).


1.1 Reagents


(1) RNase Out 40U/ul (Invitrogen, 5000U, Cat. No. 10777-019),

(2) HT7T primer, HPLC purified (Eurogentec, sequence: 5'-GGCCAGTGAATTGTAATACGACTCACTATAGGGAGGCGGTTTTTTTTTTTTTTTTTTTTTTTT-3'),

(3) Trehalose 1.7 M (Sigma, Cat. No. T-5251),

(4) 5 x first strand buffer (SuperScript II RT, Invitrogen, Cat. No. 18064-014),

(5) 0.1 M DTT (SuperScript II RT, Invitrogen, Cat. No. 18064-014),

(6) 10 mM dNTP-mix (Amersham Pharmacia Biotech, 100 mM, Cat. No. 27-2035-01),

(7) SuperScript II RT 200U/ul (Invitrogen, 10000U, Cat. No. 18064-014),

(8) 5 x second strand buffer: 100 mM Tris-HCl (pH 6.9) (Invitrogen, Cat. No. 15714-025 (Tris) and 1027 (HCl)), 450 mM KCl (Sigma, Cat. No. P-9541), 23 mM MgCl2 (Sigma, Cat. No. M 8266), 0.75 mM Beta-NAD+ (Sigma, Cat. No. N-1636), 50 mM (NH4)2SO4 (Invitrogen, Cat. No. 15501-018)),

(9) E. coli DNA Ligase 10U/ul (Invitrogen, 100U, Cat. No. 18052-019),

(10) E. coli DNA Polymerase I 10U/ul (Invitrogen, 1000U, Cat. No. 18010-025),

(11) E. coli RNase H 2U/ul (Invitrogen, 120U, Cat. No. 18021-071),

(12) DEPC-water (Invitrogen, Cat. No. 10977-015),

(13) Ethanol Absolut (VWR, Cat. No. 1.08543.0250),

(14) QIAquick PCR Purification Kit (Qiagen, Cat. No 28106),

(15) NaAc 3M, pH 5.0 (Sigma, Cat. No. S-2889),

(16) AmpliScribeTM T7 Transcription Kit (Epicentre Technologies, Cat. No. AS3107),

(17) RNeasy Mini Kit (Qiagen, Cat. No 74106).


1.2 Protocol First Strand Synthesis


(1) Mix 5 ug of total RNA (3 ul) with 1 ul of RNase Out 40U/ul.

(2) Add 1 ul of (HT7T) primer 200 ng/ul and 6 ul of 1.7 M Trehalose heated to 42C.

(3) Put on 75C for 5 minutes, chill on ice and spin down.

(4) Add 4 ul of 5 x first strand buffer, 2 ul of 0.1 M DTT, 1 ul of 10mM dNTP-mix, 1 ul of 1.7 M Trehalose and 1 ul of SuperScript II RT.

(5) Incubate at 37C for 5 minutes, at 45C for 5 minutes and then make 10 cycles of 60C for 2 minutes and 55C for 2 minutes. Go to 65C for 10 minutes and then go to 4C or chill on ice.


1.3 Protocol Second Strand Synthesis:


(1) Add 33.4 ul of 5 x second strand buffer, 3.4 ul of 10 mM dNTP-mix, 1 ul of E. coli DNA Ligase (10U/ul), 4 ul of E. coli DNA Polymerase I (10U/ul), 1 ul of E. coli RNase H (2U/ul) and 103.8 ul of DEPC-water to the 20 ul of first strand mix.

(2) Incubate at 16C for 3 hours, at 65C for 10 minutes and then go to 4C or chill on ice.

(3) Purification with QIAquick PCR Purification Kit according to the manufacturers guidelines.



1.4 Protocol for In Vitro Transcription (IVT):


(1) Add 2 ul of 10 x T7 reaction buffer (heated to 42C), 6 ul of 25 mM rNTP-mix, 2 ul of 100 mM DTT and 2 ul of AmpliScribe T7 enzyme solution to the DNA from second strand synthesis.

(2) Incubate at 37C for 3 hours, at 65C for 10 minutes and then go to 4C or chill on ice.

(3) Purification with QIAgen RNeasy Mini Kit according to the manufacturers guidelines.

(4) Check RNA concentration by OD. We recommend a 1/10 dilution in water or no dilution when using Nanodrop. Check A260/A280 ratio and determine the RNA concentration (1 OD = 40 ng/ul).


2 Cy5 Labeling



2.1 Reagents:


(1) RNase Out 40U/ul (Invitrogen, 5000U, Cat. No. 10777-019),

(2) Random nonamers N9 1 ug/ul (Genset, 1862270),

(3) 5 x first strand buffer (SuperScript II RT, Invitrogen, Cat. No. 18064-014),

(4) 0.1 M DTT (SuperScript II RT, Invitrogen, Cat. No. 18064-014),

(5) 10 mM dNTP/2 mM dCTP (Amersham Pharmacia Biotech, 100 mM, Cat. No. 272050/60/70/80),

(6) CyTM-3-dCTP and CyTM-5-dCTP for microarrays (Amersham Pharmacia Biotech, 25 nmol, Cat. No. PA 53021 and PA 55021),

(7) SuperScript II RT 200U/ul (Invitrogen, 10000U, Cat. No. 18064-014),

(8) 2.5 M NaOH (Sigma, Cat. No. S-0899),

(9) MOPS (3-(N-Morpholino)propanesulfonic acid) (Sigma, Cat. No. M-8899),

(10) NaAc 3M, pH 5.0 (Sigma, Cat. No. S-2889),

(11) QIAquick PCR Purification Kit (Qiagen, Cat. No 28106).



2.2 Cy5 Labeling Protocol:


(1) Mix 5 ug of RNA from IVT (8 ul), 0.5 ul of RNase Out 40U/ul, 5 ul spikes (not for Arabidopsis thaliana) and 2 ul of random nonamers N9 1 ug/ul.

(2) Denaturate at 70C for 10 minutes, chill on ice and add the following components: 4 ul of first strand buffer, 1 ul of 10 mM dNTP/2 mM dCTP, 2 ul of 0.1 M DTT, 1.5 ul of Cy5 dCTP and 1 ul of SuperScript II RT 200U/ul.

(3) Incubate at 42C for 2.5 hours and then go to 4C or chill on ice.

(4) Denaturate the RNA template by adding 2 ul of 2.5 M NaOH, incubating at 37C for exactly 15 minutes, adding 10 ul of 2 M MOPS and putting on ice.

(5) Purification with QIAquick PCR Purification Kit according to manufacturers guidelines.

Parameters
Amount of nucleic acid labeled, Amplification, Label used
Links
All 14 experiments using protocol P-MEXP-579