( f an insufficient amount of DNase I. To...)Determinants of RNA Integrity and Purity

Figure 2. Different Types of Genomic DNA Contamination In Total RNA Preparations. Agilent 2100 bioanalyzer electropherograms of RNA contaminated with high molecular weight genomic DNA (A), partially sheared genomic DNA (B), and small DNA fragments generated by incomplete DNase I digestion (C).

Small RNAs, such as 5S and tRNAs, are efficiently recovered in organic extraction methods but are depleted in column-based purification methods (compare Panels A and C, and Panels B and D in Figure 1). At Ambion, we have found that total RNA samples in which small RNAs have been removed by solid phase extraction (following organic extraction) show superior performance in RNA amplification compared to RNA isolated by organic extraction only. Since small RNAs can comprise as much as 15% of total RNA, their removal effectively increases the percent of mRNA within the total RNA sample and decreases the potential for interference during cDNA synthesis.

The rRNA Ratio Should Not Be Used as the Sole Indicator of mRNA Quality
The 28S:18S rRNA ratio has traditionally been viewed as the primary indicator of RNA quality, with a ratio of 2.0
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