Use this category for the exchange of ideas, methodologies and references regarding the isolation, manipulation and analysis of RNA. (Extraction protocols, Northern Blot analysis, RNase Protection, Differential Display, In Vitro Transcription, etc.)
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Technically is there any problem with Northern blot for long transcripts, let say over 7 kp? Do we have to do some sort of treatment like when we do Southern for large DNA fragments (HCl treatment)?
We have cloned full sequences of 4 genes and now we wanna study their expression. But we have failed to obtain any signal from all of these genes by Northern blot. Their sizes are around 7 to 10 kb and their mRNA sizes are approx. 6 to 9 kb.
What you are really interested in is what you often gossip about (F.Crick)
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I haven't done Northerns of long transcripts before, but I'm curious, have you confirmed their expression by qPCR?
Also, a quick check on Ji, et. al., (2003) showed that they were able to detect MALAT-1 (~8kb) on a northern, and don't seem to use anything special.
"..10 mg of (total RNA) was subjected to electrophoresis and Northern blotting. MALAT-1 was hybridized with a digoxygenin-labeled RNA probe of 800 nt cloned from the RACE reaction for MALAT-1.."
You could try remaking your probes to have multiple concatenated target binding sites? (source: hearsay, it apparently improved signal multi-fold)
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I don't remember details, but you might check whether a lower % gel might transfer better.
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