( Fig.1 In Figu...)New Kit Generates Exceptionally Pure Total RNA for RT-PCR

Fig.1

In Figure 1, PCR analysis was performed to demonstrate the absence of DNA contamination in RNA isolated from different quantities of mouse tissue and cells. Using the Absolutely RNA kit, total RNA was isolated from mouse liver and mouse NIH/3T3 cells. From 5, 20, and 40 mg of liver, RNA yields were 14, 37, and 100 g, respectively, and from 10⁵, 10⁶, and 10⁷ cells, RNA yields were 2, 19, and 142 g, respectively. Using Stratagenes prostar first strand RT-PCR kit³, cDNA was synthesized, and control reactions were run in the absence of reverse transcriptase. A 1-l aliquot of each of these reactions was used in PCR (Methods). The PCR included primers specific for glyceraldehyde-3-phosphate dehydrogenase (GAPDH) related sequences that are present in greater than 200 copies in the mouse and rat genomes.⁴ The generation of a 0.2-kb PCR product from the control samples lacking reverse transcriptase (RT) indicates that DNA is present in the RNA sample. The absence of PCR product in the RT minus samples demonstrates that DNA is not detectable in the RNA purified with the Absolutely RNA kit.

RNA Samples Isolated Using Various Kits

Total RNA was isolated from tissue and cultured cells using several commercial kits, then examined by RT-PCR to determine which samples contained DNA contamination. RNA was isolated using Ambions RNAqueous kit, Clontechs NucleoSpin RNA kit, Qiagens RNeasy kit and Stratagenes Absolutely RNA RT-PCR miniprep kit, according to the instructions provided for each. An additional kit (Boehringer Mannheims High Pure RNA Isolation k
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