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 d(T)19N||2 mg/ml||1|
|pd(N)6 or 9||2 mg/ml||1|
|RNA (> 75 µg/ml)||1 to 3 µg||13.5|
|RT Mix||[ ]||µl|
|SuperScript II||200 U/µl||1.9|
|Incubate:||10 min. at 65 °C|
|Neutralize:||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. Alternatively, the reaction can be cleaned up using a protocol for Zymo columns, which has the benefit of very small elution volumes.
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 one 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). The precise colume you need will depend on the size of your cover slip - adjust volumes acordingly.
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||40x||Stock [ ]||Volume|
|dATP||500 µM||20 mM||100 mM||10 µL|
|dTTP||300 µM||12 mM||100 mM||6 µL|
||8 mM||50 mM||8 µL
|5-(3-aminoallyl)-2'-deoxyuridine 5'-triphosphate||Ambion||8439||50ul/50mM Soln, $100||This is the best deal.|
|5-(3-aminoallyl)-2'-deoxyuridine 5'-triphosphate||Sigma||A0410||going up and up||Forget Sigma, buy Ambion.|
|Cy3 + Cy5 Mono-Reactive dye pack||Amersham||RPN5661||$248.00||12 tubes each Cy3 & Cy5. Each tube can be used for 1-3 rxns. No aliquoting necessary.|
|Cy3 Mono-Reactive dye pack||Amersham||PA23001||$170.00||5 tubes, 12-16 rxns each.|
|Cy5 Mono-Reactive dye pack||Amersham||PA25001||$170.00||5 tubes, 12-16 rxns each.|
|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:
notes: These mono reactive esters are labile in water, and susceptible to moisture. DMSO is used as an aliquoting reagent to avoid this problem, but stability of the aliquots can still be a problem. This aliquoting method is only used for the larger dye packs (PA23001 & PA25001). Only make aliquots if you don't plan on using an entire tube all at once (i.e. 12-16 rxns). Otherwise it's better to use the smaller dye packs (RPN5661) and avoid making aliquots altogether.
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.