Reverse+transcription+using+random+pentadecamer+primers+increases+yield+and+quality+of+resulting+cDNA

Reverse transcription using random pentadecamer primers increases yield and quality of resulting cDNA http://www.biotechniques.com/BiotechniquesJournal/2006/May/Reverse-transcription-using-random-pentadecamer-primers-increases-yield-and-quality-of-resulting-cDNA/biotechniques-45710.html Michael Stangegaard1, Inge Høgh Dufva2, and Martin Dufva1 BioTechniques 40:649-657 (May 2006) doi 10.2144/000112153 pmid 16708763

paper originally found by Mara

"C:\kurt\storage\CIM Research Folder\DR\2013\2-12-13\Reverse transcription using random pentadecamer primers increases yield and quality of resulting cDNA.pdf"

Notes 2-13-12

Reverse transcription using random pentadecamer primers increases yield and quality of resulting cDNA

haven't heard of amplified mRNA aRNA)

q random hexamers induced reverse transcription of only 40% of the template aRNA

q random pentadecamer-primed reactions resulted in 11-fold more genes being detected in whole transcriptome DNA microarray experiments than random hexamer-primed reactions

q Amplification was performed with the RiboAmp® T7-based RNA amplification kit

q Slides prespotted with 44,290 60-nucleotide-long oligonucleotides directed against the human transcriptome were obtained (Agilent Technologies), and hybridizations were performed according to supplier’s instructions.

q The data was normalized using the Qspline normalization method (25), and further analysis was performed using Excel®.

q There was no difference in size distributions of the respective cDNA populations produced by different primers.

pentadecamer primers yielded more cDNA than hexamers with all the reverse transcriptases they tried

Taken together, the results showed that R15 priming increases the amount of the resulting cDNA, but also yields cDNA with better representation of the original mRNA population.

q background signal, calculated as the average of the 1000 weakest signals on the array.

q It is not clear why the use of R15 primers results in an increased yield of cDNA compared with R6 primers.

they think that shorter primers may have difficulty annealing

q It is therefore likely that R15 or longer primers will bind better to ATrich sequences than R6 primers during the reverse transcription procedure, indicating a mechanism for the observed increase in transcriptome coverage using longer primers.

q The lower yields obtained using random primers that were 18 nucleotides or longer can be the result of secondary structure formation or increased specificity resulting in fewer binding sites for the primers.

q balanced between melting temperature of the primer-template duplex and the increased specificity originating from the increasing length.

q Techniques liker laser capture microdissection (LCM) enable the isolation of single cells

q In conclusion, a simple substitution of random hexamers with random pentadecamers in the reverse transcription reaction has many advantages, such as better genome coverage and a better yield.