We use 1 ug RNA for cDNA synthesis and work lung, prostate, lymph and primer tumor tissues.
there is a huge difference between oligodT and randomX:
oligodT; advantages:
- mature, eucaryota mRNA and viral during activation
- no bacterial mRNA background (perfect for colon cancer)
- perfect for low-copy genes since rRNA is ommited (80-95% of total RNA)
- high (ca. 40-45C) RT temperature
disadvantages:
- 18s rRNA cannot be assessed
- 3' mRNA preference (qPCR primers must be carefully designed on long mRNA)
- low efficiency for low-quality RNA
randomX; adv:
- perfect for low-quality RNA and RNA isolated from FFPE slides (RNA fragmentation)
- 18S rRNA may be assessed
- bacterial RNA can be checked
-5' mRNA preference
- good for qPCR with probes (small amplicons)
- higher efficiency
disadvantages:
- more expensive than oligodT
- low temp of RT (hybridization at ca. 25C)
- bacterial background
- not-so good for low-copy genes
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- Science method