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What can I do to increase the banding signal? - (Jul/31/2011 )

Hi everybody!

I have some problems with my RT PCR. I am using a total mRNA isolated from spinal cord, and primers for AQP4, (with this primers I should obtain a predicted product size about 920bp – this is a full functional AQP4 sequence which was used by one of my lab mate for transfection) .
For RNA isolation I used a TRIZOL reagent, and followed the provided procedure.

RNA samples that I used for my RT PCR (measured with NanoDrop)

Sample 1: 260/280 – 2,15; 260/230 – 2,27; 1473,3ng/ul
Sample 2: 260/280 – 1,98; 260/230 – 2,08; 1174,9ng/ul
Sample 3: 260/280 – 2,18; 260/230 – 2,20; 1322,3ng/ul

I used Promega A1250 RT-PCR kit, with Beta actin primers (101bp) as control and followed the recommended conditions (40 cycles). For both primer sets the annealing temperature that were working in other experiments is 60˚C. I have got a very clear banding Beta acitn but, very weak banding for AQP4 ( I was able to see it, after sharpening and contrast adjusting on the gel image ). This weak banding was only visible in samples 2 and 3. There was no banding in sample 1.

My question is, what can I do to increase the banding signal for AQP4?

I’m quite suspicious about my RNA quality, aren’t the values at 260/280 to high? (like 2,15 or 2,18 in samples 1 and 3). Do you know what are the values if the RNA is contaminated with polysaccharides and/or proteoglycans?

Thanks for your time and consideration.

-Coffee_Boy-

Hi Coffee_Boy,

My recommendation is to re-design your AQP4 assay. While in theory your PCR should be able to amplify a 920 bp amplicon, you are making things harder than they need to. Designing a PCR assay that would amplify a fragment between 300 to 500 bp of your AQP4 gene will very likely increase your signal intensity significantly. The reason why you are getting such low PCR signals is that your amplicon is a tad too long. PCR works exponentially better the smaller the amplicon.

I would not worry about your RNA. 260/280 ratios in the 2.00 to 2.20 are perfectly acceptable and actually pretty good. If you want to determine if you have polysaccharide contamination you need to look at the 260/230 ratio, not at the 260/280 ratio (polysaccharides absorb at 230 nm). Pure RNA has a 260/230 ratio between 2.00 and 2.20. If you have polysaccharide contamination your 260/230 ratio will be lower than 2.00.

Good luck.

-ivanbio-

Sorry for being late with my reply.

Ivanbio, thanks for your answer – yes, you are right. PCR works better with smaller amplicon. However I would like to try to do it with this primers set and amplify the whole functional AQP4 sequence.

If my RNA is ok, then what can I do more? Should I use lower annealing temperature and/or increase the number of cycles? Any other suggestions?

-Coffee_Boy-

RNA is fragile and isolation may cause it to break into small pieces. So there is a possibility, that you may just have such low amount of 920bp-long intact RNA/cDNA. We, for example have actin primers creating 850 bp amplicon to check for RNA quality. On the other hand, unless you vortex your Trizol rigorously the RNA should not shear that much. Maybe the RT step can be the problem, do you use random primers or oligo dT?

You can also try to do nested PCR to increase yield, but with that comes the problem of mutation generation due to number of cycles, so proof-reading polymerase would be recommended.

-Trof-

Hi Torf, Thanks for your reply.

Trof on Wed Aug 10 17:04:06 2011 said:

So there is a possibility, that you may just have such low amount of 920bp-long intact RNA/cDNA.
Yes, this is a one thing that I had thinking about, the RNA integrity. The purity is the one thing, but it seems that the integrity (or as you wrote low amount of 920bp-long RNA) might be the cause. Do you think that the “to rigorously vortexing” during the RNA isolation steps is the main reason of its low cohesion??

Trof on Wed Aug 10 17:04:06 2011 said:

do you use random primers or oligo dT?
I have tested them both, and there was no difference between them.

I’m not sure do I have a proofreading polymerase, thus I would like to deal with it without purchasing something new.
Thanks for your time and consideration .

-Coffee_Boy-

I heard that vortexing and harsh pipetting/pushing through needle can cause longer molecules of NA to break. The column-based isolation kits also allegedly break longer molecules. But 900bp is not that big to be affected by this, AFAIK. But if you vortex the Trizol much (and other isolation steps) you may try to mix it more gently in hand, see if it changes anything.

-Trof-

hi all, i got the same problem, the problem is the gapdh positive control shows a very strong band, but not the interested gene.
i guess gapdh is quite abundant which can be picked up easily. i suggested to amplify another gene beside the housekeeping gene, and if still not able to see the band, then has to consider the possibility of the RT step.

-dandan-