Hi all, I am currently doing the northern blot using Ambion NorthenMax kit, and also use pTRI-GAPDH mouse for control probe preparation. The weird thing is the GAPDH signal is perfect but my interested genes gave no signal at all...
For my probes targeting interested genes, I used PCR generated DNA template for probe preparation, is that OK? I just directly added T7 promoter at the 5' of my sequence. All the probes are prepared using Roche DIG labeling system.
Hope you guys can give me some suggestions and advice, thank you so much.
Hello Lu
that is an acceptable way to prepare you probe
RNA Quality; that is minimal degradation of RNA is paramount for Northerns to work consistently well. Have you checked the integrity of your subunits by agarose gel or Agilent biochip ?
Its is not uncommon to yield a signal from a highly expressed housekeeping gene, even with degradation but no signal from GOI, where degradation may effect enough of your total RNA population to undermine detectable signal. In contrast your housekeeper is so highly expressed that even with significant degradation enough of your RNA may be undegraded to enable detection
To investigate RNA integrity:
1. Run on an Agilent biochip and look for RNA with RIN numbers > 8.0
2. Run on a 2% Agarose gel but first add 10% formamide to your sample and denature for 2 min @ 95C followed by snap cooling on ice
3. Run your denatured RNA on a formaldehyde agarose (that is your usual Northern) gel
Thank you Laurence! You are really helpful. The thing is I checked my RNA quality with Bioanalyzer, all the samples gave a RIN around 9.0-9.5, which means good RNA integrity. Now I am concerning the DIG-label efficiency and the expression level of my GOI for successful detection.
Yes, you are right, Thank you Andrei. I saw my 5'-probe labeling efficiency was much lower than GAPDH. Is this possible due to my 5'-Probe was generated by PCR but GAPDH probe was generated by digested plasmid? And why it is a bad idea to load probes together with samples on the same northern gel ?
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