( ion factor 1 a) medium-abu...)New Kit Generates Exceptionally Pure Total RNA for RT-PCR

ion factor 1a), medium-abundance transcripts (b2-microglobulin), and low-abundance transcripts (protein phosphatase 1 and ornithine decarboxylase). Although b2-microglobulin mRNA is classified as medium abundance, its expression level is higher in liver than in brain⁷ (Figure 3). Similarly, ornithine decarboxylase is expressed at overall low levels but the levels vary in tissues throughout the body.⁸ The presence of the expected PCR bands in Figure 3 shows that even rare RNA transcripts are represented in total RNA isolated with the Absolutely RNA kit.

Conclusions

RNA isolated with Stratagenes Absolutely RNA RT-PCR miniprep kit is ideal for RT-PCR. The kits quick, convenient protocol includes a DNA removal step that ensures sensitive RT-PCR results without having amplification products generated from genomic DNA targets. Moreover, RT-PCR readily detects low-abundance transcripts from this RNA. The Absolutely RNA RT-PCR miniprep kit effectively generates high-purity, intact total RNA from a range of tissues and cultured cells, ready for the most sensitive RT-PCR experiments.²

Methods

The ProSTAR first strand RT-PCR kit was used for cDNA synthesis.³ For GAPDH primer detection of DNA in total RNA samples, control cDNA synthesis reactions were performed in the absence of reverse transcriptase. PCR reactions contained 1 l from the cDNA reactions (100 ng of total RNA), 200 M of each dNTP, 25 ng of each GAPDH primer, 1X Taq polymerase buffer, and 1.25 U of Taq2000 DNA polymerase. PCR react
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