This month's questions from the Molecular Biology Forums (online at http://molecularbiology.forums.biotechniques.com) comes from the “Microarrays” section. Entries have been edited for concision and clarity. Mentions of specific products and manufacturers have been retained from the original posts, but they do not represent endorsements by, or the opinions of, BioTechniques.
Molecular Biology Techniques Q&A How can I improve the incorporation of my CyDye? (Thread 15987)Q I have been trying to CyDye-label a difficult batch of RNA samples using an indirect aa-dUTP method. I ran the RNA on an agarose gel to check for degradation and the quality seemed good. The spectrophotometer readings were also good, with a 260 nm reading of less than 1.0 and 260/230 and 260/280 ratios higher than 2. These samples were extracted using the Tri-reagent method and further purified with Qiagen's RNeasy cleanup kit.
Using the Invitrogen Superscript II kit, I reverse-transcribed 10 µg RNA with seven different aa-dUTP/dTTP ratios (1:4, 3:7, 2:3, 1:1, 3:2, 7:3, and 4:1) with Ambion aa-dUTP labeling. I hydrolyzed and neutralized the RNA with NaOH and HEPES, and then used a Qiagen column to purify the cDNA. I replaced the standard Qiagen buffers with non-Tris– containing buffers and eluted the cDNA from the column using a phosphate elution buffer. According to the 260-nm reading, this process yielded about 1.5 µg cDNA. I dried the resulting pellet at 60°C in a SpeedVac and re-suspended it in 3 µL water followed by 1.5 µL 0.3 M sodium bicarbonate at pH 9. I then mixed it with 4.5 µL CyDye in DMSO and incubated it overnight in the dark. (Incubation for 1 h is normally recommended, but I extended it since the longer incubation time results in slightly better incorporation.) I purified the label with Qiagen columns using the standard buffers. I measured the incorporation and FOI using the spectrophotometer at 260 nm and 550/650 nm.
The results were terrible. The incorporated dye only averaged 17 pmols and the FOI averaged 10. The cDNA quantity was an average of 630 ng although this varied with the aa-dUTP/ dTTP ratio.
How can I improve the incorporation and FOI to a level where I can hybridize?
A Are you sure that you solubilized all of your cDNA prior to labeling?
A Have you considered direct labeling? There is a labeling technology called the Universal Linkage System (ULS) from Kreatech Diagnostics. The ULS molecule forms a coordinative bond with the N7 position of guanine in your RNA. The reaction takes about 15 min. The ULS is available coupled to a wide variety of labels including CyDyes. If you need any more information, you can visit the company's web site.
A Direct labeling works really well. I use it myself and it's quick and easy. You can just look it up online since there is plenty of information for those who are interested.
A Depending on the dye and the DNA polymerase or reverse transcriptase, direct labeling can work very well. Most DNA polymerases incorporate Rhodamine dye set labels very well. If that won't work with the excitation and emission characteristics of your detection system, you might want to contact Dr. Thomas Myers for mutant thermostable DNA polymerases and reverse transcriptases that incorporate cyanine dye–labeled dNTPs very well.
A I don't know whether FOI means the fraction of reagent incorporated or the fraction of target amines modified. But whichever you mean, an FOI of 10% doesn't necessarily mean that the reaction didn't work. The degree of modification at completion of an amine modification reaction is a balance between two competing reactions: amine modification (the desired reaction) and hydrolysis (a non-productive consumption of reagents). The expected outcome of this competition depends crucially on the molar concentrations of both the reagent and the target amine, not just on their molar ratio. I wrote a paper on this subject (Bioconjugate Chemistry 2006. 17:501-506). Here are some practical tips:
The buffer must not contain competing nucleophiles such as primary and secondary amines (e.g., Tris) and sulfhydryl groups (β-mercaptoethanol or dithiothreitol).
Allow the reaction to go to completion. This will take at least several hours. Overnight incubation might be preferable. This will help prevent you from wasting your expensive reagents.
Large dyes can't be effectively dialyzed. Your protocol likely calls for ethanol precipitation, which is probably fine.
Q I have been trying to incorporate amino-allyl dUTP via PCR for dye conjugation with Alexafluors 555 and 647. The protocol recommends a 3:2 (aa-dUTP to dTTP) ratio in the PCR reaction mix, but when I check the actual incorporation (base/dye) it's quite high with only about one fluoro-label every 140 bp.
I tried increasing the ratio of aa-dUTP to dTTP to increase the dye incorporation and found that 4:1 was a good ratio. It returned one fluoro-label every 60 bases or so and did not inhibit the PCR.
However, when I used this 4:1 sample for hybridization on my chip, the signal was very weak. Weaker, in fact, than when I used the original 3:2 ratio with slightly more DNA.
So my question is whether the amino-allyl group on the dUTP interferes with hybridization if there are too many of them. Will one fluoro-label every 140 bp work for indirect labeling, if I use the originally recommended 3:2 ratio?
A I use a 7:3 ratio; it works great for me.
Q Did you check how many labels are incorporated (base/dye) for the 7:3 ratio?
I am just concerned that my incorporation (140 bp/dye) is worse than what should be expected from a 3:2 ratio.
A Go to this web page: www.pangloss.com/seidel/Protocols/probecheck.html. Perhaps this information will help.
