1. I silenced a gene by RNAi method, for that i chose the gene and the size of the gene is 1.5kb and my insert size is 500nts. i did the transfection and i want to confirm the silencing by northern analysis. when i did i got the ribosomal RNA instead of my specific mRNA reduction and dsRNA accumulation.

  2. Total RNA was prepared with TRIzol reagent (Sigma), and 20 μg/lane was fractionated on a 1.2% agarose, 2.2 M formaldehyde gel. The RNA was visualized with ethidium bromide. The RNA was transferred to a nylon membrane (Hybond; Amersham Biosciences) for 14 hrs and probed with [α-32P] UTP-labeled used SSC for transfer and Prehybridization at 55 for 2hrs incubation, in the probe for 10hrs and Washing at 60 for 2X SSC 20 mins two times.

In this image you can the samples TET induced and uninduced RNA. but the result was confusing and am getting such a strong ribosomal RNA (2.2 Kb) contamination.

  • $\begingroup$ Did you try a dot blot or northern with your pure dsRNA to check if the probe is even working? You can use IVT to produce it. You can even use your plasmid as a proxy for dsRNA for dot blot. How big is the probe? $\endgroup$ – WYSIWYG Mar 28 '19 at 23:48
  • $\begingroup$ Not really a constructive comment but nowadays hardly anyone uses northern blot. qRTPCR is commonly used for quantification of RNA: it is fast, clean and sensitive. $\endgroup$ – WYSIWYG Mar 28 '19 at 23:52
  • $\begingroup$ Yes, i did IVT to synthesis my probe, its 500nts, in our lab we don't to qRTpcr, we believe in basic biology, i put my insert in tetracycline inducible plasmid..when i induce the cells with TET the dsRNA synthesis and it blocks mRNA production. my question is there any way to reduce the rRNA...if i reduce my rRNA i can get my mRNA specific band, any suggestion? $\endgroup$ – PraveenKumar Mar 29 '19 at 8:22
  • $\begingroup$ Since your probe is 500nt long, you can increase the hybridization temperature. You can increase the washes too. You can also check if your probe has any complementarity with rRNAs. If so, you may design a better probe. By dot blot, I meant doing a blot with pure RNA samples instead of total RNA. BTW, you may like and prefer to do northerns for whatever reason but don't justify by saying that it is "basic biology" as if qPCR is something that belongs somewhere else. $\endgroup$ – WYSIWYG Mar 31 '19 at 10:25
  • $\begingroup$ Thank you for your suggestion i will do that. sorry for that, I mean to say the qPCR enzymes are too costly as compare to alpha-UTP.. $\endgroup$ – PraveenKumar Mar 31 '19 at 13:27

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