The following protocol describes how to make Cy3 and Cy5 labelled fluorescent probes from RNA samples using reverse transcription and an amino-allyl modified nucleotide for esterification of the Cyanine dyes. It is optimal for yeast RNA and may need modification with other RNAs.
The first step is to reverse transcribe the RNA into cDNA in the presence of the amino-allyl nucleotide. Then we stop the reaction by base hydrolysis of the RNA. After neutralization and clean up the cDNA is allowed to conjugate to the reactive Cy dyes. Once that reaction is quenched and cleaned up, the probe is ready to go.
|Oligo dT or pd(N)6||5 mg/ml||1|
|Volume of RNA||1 to 2 µg||14.5|
|RT Mix||[ ]||µl|
|SuperScript II||200 U/µl||1.9|
10 µl 1N NaOH
10 µl 0.5M EDTA
|Incubate:||15 min. at 65 °C|
|Neutralize:||25 µl 1M Tris ph 7.4, or 25 µl 1M HEPES pH 7.0|
The Qiagen PCR clean up kit works well for this step. However it is important to keep in mind the DNA binding curve for silica, on which this kit is based, is favorable at low pH but falls off precipitously around pH 8. Thus it is essential that the pH of your reaction be below 7.5 by the time it hits the Qiagen membrane.
Protocol for Qiagen PCR clean-up kit:
Eluted volume is typically around 48 µl. I elute with 1/2x PE buffer and reduce the volume for hybridization in the spin vac.
You can check your probe by scanning it in a spectrophotometer (200-700nM). You should see three peaks of absorbance. One at 260 nM for the cDNA, and then on each at 550 and 650 nM for Cy3 and Cy5 respectively.
Combined reactions should be in a volume of 18 µl (can use spin vac to control volume or dry down and resuspend in H2O).
3.6 µl 20X SSC
1.8 µl polyA (10 mg/ml)
0.54 µl 10% SDS
|denature:||2 min. at 95 °C|
The probe is now ready for slide hybridization.
|dNTP||1x||50x||Stock [ ]||Volume|
|dATP||500 µM||25 mM||100 mM||10 µL|
|dTTP||300 µM||15 mM||100 mM||6 µL|
||10 mM||100 mM||4 µL
|5-(3-aminoallyl)-2'-deoxyuridine 5'-triphosphate||Sigma||A0410||5 mg for $277.40|
|Cy3 Mono-Reactive dye pack||Amersham||PA23001||$170.00|
|Cy5 Mono-Reactive dye pack||Amersham||PA25001||$170.00|
|Hydroxylamine (50% wt soln in H20) FW 33.03
(also available as HCl salt from Sigma H9876 100g/$10.40)
Prepare aliquots of Cy dyes as follows:
*note: Dioxane was suggested as an alternative to DMSO, which takes a long time to dry. However it appears that Cy5 is not soluble in dioxane. Previously H2O was used, but the mono reactive ester is labile in water.
The extinction co-efficient for the amino-allyl dUTP in 0.1 M PO4, pH 7.5 is:
dNTP Calculator - Calculates the concentration of aa-dUTP when the absorbance is entered.