Hello
I did several times to detect the miRNA by northern blot.
Briefly explain my protocol.
Small RNA was prepared by extraction kit and load the 2.5ug of RNA on 15% TBE-urea gel 1hour. After transfer, membrane was UV-cross linked and baked at 60℃.
Pre-hybridized the dried membrane 3h and then hybridized overnight with DNA oligonucleotides complementary to miRNA or RNA U6 that had been 3’ end labeled with biotin.
Blocking and streptavidin-HRP (1:1500) reaction 30m each and did ECL (pico detector, Applichem) 5m and acquired the image with a CCD camera (LAS-4000)
RNA_U6 and marker was detected at few minutes, but miRNA was not detected even exposure 30m.
My question is exposure time (30m) is too short? Then what is the appropriate miRNA detection exposure time ?
And the ECL reagent is not enough to detect the miRNA?
Thank you for reading :)
Matthias R. Schaefer
When I did a real time PCR to know the expression level of Target miRNA, CT value of RNA_U6 was about 8 and target miRNA was 15. (Synthesis 500ng of cDNA)
Is it low expression level ?
Now I know Biotin label probe is uncommon way to detect miRNA
Of course at first time I considered DIG-labeled RNA probe, But In our lab, miRNA detection by northern blot is first time.
So I used biotin labeled DNA probe to limited the variable.
I will change the probe to 3’ tail of DIG label and will try again.
Thank you very much :)
Hi Kwak,
I think 2.5 ug is not enough for miRNA northern blotting, unless you are trying to detect very abundant miRNAs, such as miR-21 (in the case of mammalian cells). Although the amount of total RNA to be used depends on the abundance of your miRNA and should be determined empirically, I think 10-30ug would be a good starting point. Note that, assuming comparable amplification efficiency (I know this is a lousy assumption), your miRNA is expressed at level ~128-fold lower than U6, which means that you should increase the sensitivity more than 100-fold to achieve decent signals for your miRNA. So, increase the RNA amount, make sure that your probe is labeled with high specific activity (Schaefer made a good point), and increase the expose time.
Dooyoung Lee
Thank. You for your advice. I tried again with 10ng and 20ng of small RNA, unfortunately miRNA was not detected. I am not sure some researcher said biotin probe also enough to detect miRNA, in my case it was failed. As mentioned above, I will change my probe to dig tailing. If I got a result I will update that :)
Hi Kwak,
I am also interested in the Northern application for small RNAs and I was thinking to use biotin-probes as suggested here: https://www.ncbi.nlm.nih.gov/pubmed/?term=Liquid+Hybridization+and+Solid+Phase+Detection%3A+A+Highly+Sensitive+and+Accurate+Strategy+for+MicroRNA+Detection+in+Plants+and+Animals
In fact, they suggest biotin-tailled as better probes that DIG labelling... With your experience, I am really doubting that now.
Have you finally tried the DIG ones?
Ana Gámez-Valero
I got dig tailing kit last week. I will try it several days after.
After that, I will share my result. thank you.
Ana Gámez-Valero
I did northern blot using dig tailing kit and share my result.
I load 30ug of Total RNA on 15% urea page gel.
Left side is the test of dig tailing probe sensitivity, and right side is northern blot.
in my case, I got a band by dig tailing kit.
if I have some time to do biotin probe sensitivity test, I will share that also later.
then we can compare two probe clearly.
Junsoung Kwak
Hi!
I just started now my experiments with biotin-labelled probe but not positive result to date. I am doubting about the hybridization temperature and the possible crosslinking method. Some papers said it should be 10ºC below the Tm and others suggest around 30º.... For the concentration, would you follow the indications of the manufacturer or the estimated concentration in others works?
Now, I have followed the same protocol I shared with you but I don't get it.
How did they go your biotin-experiments?
Ana Gámez-Valero
Sorry for late.
About hybridization temperature, in my case, I used PerfectHyb hybridization solution so that followed the manufactural protocol. (And also probe concentration is explained)
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