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Technical support : Labeling 1/2
1. Background informations :
This is the two-step method at the microarray center to generate labeled cDNA from poly(A) RNA. In the first step, amino allyl dUTP (AA-dUTP) is incorporated during reverse transcription. In the subsequent step, monofunctional forms of Alexa fluor 555 and Alexa fluor 647 dyes react chemically with the amine-modified cDNA.
The method was initially described by Randolph and Waggoner (Nucl Acids Res, 1997, 25, 2923), and has emerged as the most popular approach for the field of microarrays. The method typically incorporates higher levels of label than the direct labeling of ss cDNA (single-step incorporation of fluorochrome-dUTP), because reverse transcriptases are less efficient in using bulky, tagged nucleotides as substrate. Contrary to enzymatic labeling, the indirect method results in an even incorporation of both dyes into cDNA. A range of esterified dyes are now available that can replace the common cyanine dyes in cDNA labeling procedure.
Use poly(A) RNA as far as possible (0,65-1,3 µg/cm2) that gives best results in labeling and hybridisation from our experience. The minimum amount of total RNA which is needed for a microarray hybridisation experiment is 20 µg. If the amount of starting material is even more limited, it will be necessary to amplify the RNA. The procedure has been modified from various protocols found in the literature, and is suitable for labeling of RNA extracted from mammalian cells. Modifications should be considered for samples with different origins since the GC nucleotide content and the complexity of the RNA populations may be notably different.
2. Material
5-(3-aminoallyl)-2' deoxyuridine-5'-triphosphate (AA-dUTP) (Sigma, cat#A0410)
100 mM dNTP Set PCR grade (Invitrogen, cat#10297-018)
Random hexamer primers (Invitrogen, cat# 48190-011)
Oligo(dT)12-18 primers (Invitrogen, cat# 18418-012)
Superscript II reverse transcriptase (Invitrogen, cat# 18064-014)
RNase OUT (Invitrogen, cat# 10777-019)
Alexa fluor 555 and 647 reactive dye decapacks (Invitrogen, cat# A32755)
QIAquick PCR purification kit (Qiagen, cat# 28106)
3. Reagent preparation
3.1. Labeling mix
Prepare a 100 mM dUTP stock solution in DEPC-treated water. Vortex to mix, and transfer aliquots into new centrifuge tubes and store at -20°C.
50x labeling mix with 2:3 AA-dUTP:dTTP ratio is prepared by mixing the following reagents :
Store unused aliquots at -20°C.
3.2. Sodium carbonate buffer
Dissolve 10.8 g Na2CO3 in 80 mL of MilliQ water and adjust pH to 9.0 with 12 N HCl. Bring up the volume to 100 mL with MilliQ water.
Carbonate buffer changes composition over time : prepare it extemporaneously.
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