Optimization of Dnase I removal of contaminating DNA from RNA for use in quantitative RNA-PCR.
Reads0
Chats0
TLDR
This investigation was undertaken to optimize DNase I treatment of RNA with respect to DNA removal and mRNA preservation and found that incubation of 1 microgram RNA with 1 U of DNase for 30 min at 37 degrees C followed by heat-denaturation of the enzyme for 5 min at 75 degrees C was sufficient to destroy all the contaminating DNA, while completely preserving the respective mRNAs.Abstract:
In competitive RNA-PCR studies, contaminating DNA can produce incorrect results because of its potential to act as a second competitor. Preliminary studies using published methods for DNase I digestion of DNA as a contaminant of RNA, followed by thermal inactivation of the enzyme at 95 degrees C for 5 min before reverse transcription and PCR, suggested that the mRNA was also affected by these treatments. This investigation was undertaken to optimize DNase I treatment of RNA with respect to DNA removal and mRNA preservation. Competitive RNA-PCR of DT-diaphorase transcript was used to quantitate the effects of the various treatments. Other transcripts with varying initial concentrations were visually compared to ensure that the effects observed were not unique to specific mRNAs. With 1 U of DNase I/microgram RNA, thermal denaturation of the enzyme at 75 degrees C for 5 min preserved nearly all of the mRNA. Thermal denaturation at 95 degrees C for 5 min inactivated approximately 80% of the mRNA, whereas heating at 55 degrees C for 10 min did not completely denature the DNase I. For RNA-PCR of every transcript investigated, incubation of 1 microgram RNA with 1 U of DNase for 30 min at 37 degrees C followed by heat-denaturation of the enzyme for 5 min at 75 degrees C was sufficient to destroy all the contaminating DNA, while completely preserving the respective mRNAs. This treatment is highly recommended as a routine step in RNA-PCR and particularly with competitive RNA-PCR with human breast tissue samples (and presumably other human tissues), which are often contaminated with small amounts of genomic DNA.read more
Citations
More filters
Journal ArticleDOI
Quantification of mRNA using real-time RT-PCR
TL;DR: A series of RT-qPCR protocols are described that illustrate the essential technical steps required to generate quantitative data that are reliable and reproducible in molecular medicine, biotechnology, microbiology and diagnostics.
Journal ArticleDOI
Cloning and characterization of ADAMTS11, an aggrecanase from the ADAMTS family.
Ilgar Abbaszade,Rui-Qin Liu,Fude Yang,Stuart A. Rosenfeld,O. Harold Ross,John Link,Dawn Ellis,Micky D. Tortorella,Michael A. Pratta,Jeannine M. Hollis,Richard Wynn,J. L. Duke,Henry J. George,M.C. Hillman,Kathleen Murphy,Barbara H. Wiswall,Robert A. Copeland,Carl P. Decicco,Robert C. Bruckner,Hideaki Nagase,Yoshifumi Itoh,Robert C. Newton,Ronald L. Magolda,James M. Trzaskos,Gregory Hollis,Elizabeth C. Arner,Timothy Burn +26 more
TL;DR: A novel disintegrin metalloproteinase with thrombospondin motifs that possesses aggrecanase activity is identified and cloned, which has extensive homology to ADAMTS4 (aggrecanases-1) and the inflammation-associated gene ADAMts1, which is hypothesized to play a pivotal role in cartilage damage.
Journal Article
Possible role of valvular serotonin 5-HT(2B) receptors in the cardiopathy associated with fenfluramine.
Lawrence W. Fitzgerald,Timothy Burn,Barry S. Brown,John P. Patterson,Martha H. Corjay,Patricia A. Valentine,Jung-Hui Sun,John Link,Ilgar Abbaszade,Jeannine M. Hollis,Brian L. Largent,Paul R. Hartig,Gregory Hollis,Paul C. Meunier,Albert J. Robichaud,David Robertson +15 more
TL;DR: It is proposed that preferential stimulation of valvular 5-HT(2B) receptors by norfenfluramine, ergot drugs, or5-HT released from carcinoid tumors (with or without accompanying 5- HT(2A) receptor activation) may contribute to valvial fibroplasia in humans.
Journal Article
Characterization of human small intestinal cytochromes P-450.
Qing Yu Zhang,Deborah Dunbar,Alina Ostrowska,Stephen Zeisloft,Jiang Yang,Laurence S. Kaminsky +5 more
TL;DR: CYP3A4 is the major form of CYP expressed in human small intestine enterocytes, while CYP3A5 expression was not detected, CYP2C and, in some intestines, CyP1A1 were expressed and the highest metabolic activity occurred in the proximal intestine.
Journal ArticleDOI
The Ultimate qPCR Experiment: Producing Publication Quality, Reproducible Data the First Time
TL;DR: The critical pitfalls and sources of error in qPCR experiments are described, along with a rigorous, stepwise process to minimize variability, time, and cost in generating reproducible, publication quality data every time.