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New Kit Generates Exceptionally Pure Total RNA for RT-PCR

it) was omitted from this comparison because it is not suited for tissue samples and is limited to RNA isolation from no more than 106 cells.

Stratagenes ProSTAR first strand RT-PCR kit was used to synthesize cDNA3 and control reactions were run in the absence of reverse transcriptase. An aliquot of each of these reactions was used in PCR (Methods). RNA isolated using the kits from Ambion, Clontech, and Qiagen contained DNA contamination, as shown by the presence of a 0.2-kb PCR band in the control lanes (Figure 2). In contrast, the RNA isolated using the Absolutely RNA kit shows no evidence of DNA. This result demonstrates that the Absolutely RNA miniprep kit is effective at eliminating DNA contamination and competitive kits are not.

Fig.2

Accurate Representation

Recovery of RNA from all abundance classes is important for accurate assessment of gene transcription levels. In addition, preservation of the distribution and abundance of mRNAs is important for generating optimal cDNA libraries. RT-PCR was used to assess the presence of low-, medium-, and high-abundance classes of mRNA (corresponding to approximately 0.1% or less, 0.1 to 1%, and 1% of total RNA, respectively)5 in total RNA isolated from mouse liver and testis using the Absolutely RNA kit.

For the experiment shown in Figure 3, the ProSTAR first strand RT-PCR kit was used to synthesize cDNA from the RNA.3 The resulting cDNA was used in PCR with Stratagenes control RT-PCR primers for genes of varying abundance levels in the mouse.5,6 These include primers for high-abundance transcripts (mouse elongat
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