PCR amplification of a wide-range of fragment sizes often results in biased representation of amplified products with an increased number of short fragments.
Using a validated RNA amplification technology [ 30], large quantity of pure amplified RNA with proportional representation of source mRNA species could be generated from which cDNA could be obtained through a reverse transcription reaction.
One limitation of this RNA amplification method is the lower 5′ representation of amplified RNA due to inefficient reverse transcription at each round of the amplification step (Baugh et al. 2001; Wang 2005).
Here, using a whole genome Mycobacterium tuberculosis microarray to measure cross-genome representation of amplified mRNA populations, we have investigated two approaches to RNA amplification using different priming strategies.
To measure GCSDI across genome we combined a brief DNase I treatment of permeabilized cells with random amplification of the DNase I-nicked genomic DNA, followed with analysis of sequence representation in amplified material by a high-throughput method.
DNase I preferentially nicks DNA in open chromatin, rendering these regions inefficient template for amplification and thus predisposing them for under-representation in amplified material.
Cloning and sub-sampling individual representative amplified sequences provides a better representation of the original template amplified [21]; none of the consensus sequences generated in this study were determined from direct sequencing.
Cloning and sub-sampling individual representative amplified sequences provides a better representation of the original template amplified [ 44], therefore none of the mammoth consensus sequences generated in this study were determined from direct sequencing.
Whole genome representation of the amplified products was measured using a Mycobacterium tuberculosis microarray.
However, MDA is known to introduce a bias in the representation of individual amplified regions [ 39, 40], and a more accurate estimation must await quantitative analysis of the repeat content from flow-sorted chromosome arms DNA without the MDA step.
Although exponential amplification raises concerns about representation bias, exponentially amplified cDNA is representative of the transcript population, even when starting with the equivalent of a single cell's RNA [ 6, 7].
