Random Primers vs Oligo(dT) primers? - (Sep/06/2006 )
Í have used both random hexamers and oligodT's are two different methods to prepare cDNA for subsequent PCR reactions for cell receptor expression from cell lines. However, PCR products arising from cDNA prepared from both random hexamers and oligodT's gave different expression patterns. Is cDNA prepared from oligodT's more specific and widely used that random hexamers and why am I getting variation in expression when the only difference in the PCR reaction is the difference in cDNA preparation? Your opinions would be greatly appreciated! Thank you!
Oligo dT primers only prime at the poly-A tail of mRNAs, so this is where the reverse transcriptase starts. All cDNAs made this way will contain the 3' end of the gene. If a gene is long, the enzyme can sometimes fall off, so that the 5' end is missing. Random hexamers, though, prime randomly along the RNAs, so that the cDNAs resulting from this method represent bits and pieces of the gene along its entire length, but all regions of the gene may not be equally represented. So if your PCR amplifies a fragment near the 3' end of the gene, then the cDNA made using Oligo dT primers may indicate higher expression than if the cDNA is made from random hexamers. If your PCR fragment is near the 5' end of a long gene, then you may see an apparent higher expression in cDNA made using random primers than in cDNA made using Oligo dT primers. In other words, all regions of the gene may not be made into cDNA equally by the two methods, so that apparent PCR-generated expression levels may vary.
I also want to add that also every enzyme introduces a bias compared to the native sourch
You can check this paper out:
Properties of the Reverse Transcription Reaction
in mRNA Quantification Anders Ståhlberg, Joakim Håkansson, Xiaojie Xian, Henrik Semb, and
Mikael Kubista Clinical Chemistry 50:3 509–515 (2004)
With random hexamers the cDNA yield tend to be higher. Notice that the use of tandom 15 and 12 mer seem to further increase cDNA yield.
Check this other paper out
Reverse transcription using random pentadecamer primers increases yield and quality of resulting cDNA Michael Stangegaard, Inge Høgh Dufva, and Martin Dufva BioTechniques® May 2006
Volume 40, Number 5: pp 649-657
Bottom line: oligodt priming has bias towards the 3` end of the transcripts, random priming tend to over-represent the central part of the transcripts. None is perfect.