Dear Maryam
- if you rRNA is slightly but not totally degraded it is not uncommon to just see 1 band corresponding to 18s RNA: such RNA is obviously slightly degraded but not completely degraded as then you would just see a low Mr smear and no band at all. Such RNA can be used quite successfully for RT PCR and qPCR
- For old fashioned techniques like Northern blots and newer techniques for detecting multiple transcripts like Micro arrays you do require RNA where integrity (RIN) is high, i.e. both the 18s and 28srRNA bands are visible
- For single transcript detection in routine assays like RT PCR and qRT PCR then the presence of some degradation and thus just the 18s rRNA band is fine
- These assays generally still work because you only attempt to amplify short fragments of about 100bp and thes etend to be biased towards the 3' transcript which tends to be intact even in partially degarded RNA
Hello, Maryam Bibi,
what type of RNA are you working with? If total isolated RNA, you should see 2 distinct bands (28S and 18S ribosomal RNA for eukaryotic organisms). I may recommend to use some RNA ladder to see, whether the length of bands are correct (5.0 and 1.9 kbp).
Good luck!
Dear Maryam
- if you rRNA is slightly but not totally degraded it is not uncommon to just see 1 band corresponding to 18s RNA: such RNA is obviously slightly degraded but not completely degraded as then you would just see a low Mr smear and no band at all. Such RNA can be used quite successfully for RT PCR and qPCR
- For old fashioned techniques like Northern blots and newer techniques for detecting multiple transcripts like Micro arrays you do require RNA where integrity (RIN) is high, i.e. both the 18s and 28srRNA bands are visible
- For single transcript detection in routine assays like RT PCR and qRT PCR then the presence of some degradation and thus just the 18s rRNA band is fine
- These assays generally still work because you only attempt to amplify short fragments of about 100bp and thes etend to be biased towards the 3' transcript which tends to be intact even in partially degarded RNA
Dear Maryam
I really recommend you to use RNA ladder and make sure of what you see. furthermore, I really like using the small electrophoresis apparatus to test RNA integrity and to run the gel at low voltages (6-7 V for each 1cm of inner apparatus weight) for 35 minutes.
I saw many RNA samples that showed complete degradation only because of gel preparation and electrophoresis conditions, while it was completely fine when loaded and run carefully on well prepared gel.
good luck.
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