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I tried to make the probe several times but it failed again and again. It usually turns out that the probe after hydrolysis is very very short (maybe around 50bp). I did not check the RNA before hydrolysis except last time. However, I saw a band (though very weak) last time, but it still produce very very short probe after hydrolysis.I use Sp6 polymerase to synthesize RNA, and the original length is 1.5kb. My ideal length after hydrolysis is 500bp.

I'm guessing maybe it's the phenol contamination of the phenol:chloroform extraction step because my 260/280 value is low (around 1.6-1.7), and my 260/230 is also low, which is like 1.6-1.8. Also the peak is not at 260, it's almost at 270.

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I'm not all that familiar with in situ hybridization, but I'm missing a few details here. How did you synthesize the RNA probe (PCR, I assume?), and how long should it be? And that RNA is shorter than expected even before you use it in the in situ hybridization, do I understand that correctly? – Mad Scientist♦ Jul 16 '12 at 18:02
50bp does not sound that short for in situ... – nico Jul 16 '12 at 18:33
I had large noise with such probe, but I can see the signal. However, I need to quantify the signal so I need better probe – Pengyao Jul 16 '12 at 19:20

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Later I found out that the length of RNA read from the gel is not trustworthy. I guess it's because the RNA is in hybridyzation solution that even when it's running on the gel, it cannot reflect the actually length of the probe. The only way to test the probe quality is to do the in situ.

For the pheno contamination problem, I think it's a separate problem of myself. Once RNA can be synthesized in vitro, it means there should not be pheno contamination.

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