The signal varies with physiological state and RNA in situ signals are present when the transcription is quite high.
The probes are all working and the protocol is fully standardized
Is it so that the low abundance of transcripts is the reason for failure?
you already replied to your question :) is as you said: Low abundance of transcript make the in situ detection difficult. you can try with signal amplification protocols to increase the in situ signal...best of luck!
For in situ PCR I recomend you an ethanol-acid fixation of the brains in chilled Ice and then mounted in paraffin as usually. This fixation preserves the mRNA that could be treated with pepsin and prior the Reverse Transcription and in situ PCR. Good luck,
Indeed PCR is a much more sensitive detection methodology than RNA in situ and hence will give positive results even when the latter is negative. I would agree with Martin if you would want to add an in situ PCR or as Fulvio suggested a signal amplification protocol to increase the signal. Given all the constrains I still see PCR as a much more robust technique for detection of low abundance RNA. However make sure that your PCR primers span across exons so that you are looking at completely spliced RNAs in your in situs and the same signal doesnt come using primers (giving amplicons of similar molecular weight) that span between an exon and adjacent intron (negative control).
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