This protocol describes how to perform qPCR of genomic DNA in order to assess whether there is DNA contamination in purified extracellular RNA samples.
Method Article
qPCR of Genomic DNA to assess DNA contamination of extracellular RNA samples
https://doi.org/10.1038/protex.2015.118
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This protocol describes how to perform qPCR of genomic DNA in order to assess whether there is DNA contamination in purified extracellular RNA samples.
Extracellular RNAs (exRNAs) have been identified in every biofluid that has been tested. They have been found in extracellular vesicles, ribonucleoprotein complexes and lipoprotein complexes. exRNAs are interesting because they may serve as signalling molecules between cells, they have the potential to serve as biomarkers for prediction and diagnosis of disease, and exRNAs or the extracellular particles that carry them might be used for therapeutic purposes.
The Sample and Assay Standards Working Group of the Extracellular RNA Communication Consortium (ERCC) is a group of laboratories funded by the U.S. National Institutes of Health to develop robust and standardized methods for collecting and processing of biofluids, separating different types of exRNA-containing particles and isolating and analyzing exRNAs. In our first joint endeavour, we held a series of conference calls and in-person meetings to survey the methods used among our members, placed them in the context of the current literature and used our findings to identify areas in which the identification of robust methodologies would promote rapid advancements in the exRNA field.
A full list of the protocols developed during this effort is available at the exRNA Portal, the ERCC's website ( "http://exrna.org/resources/protocols/":http://exrna.org/resources/protocols/ ). This protocol for qPCR of genomic DNA is a quality control step in the extracellular RNA purification process.
ValidPrime kit (Human, FAM) (TATAA Biocenter, catalog # A105P25)
TaqMan 2X Universal PCR Master Mix, no AmpErase UNG (Life Technologies, catalog # 4324018)
qPCR Machine
96-well plates compatible with qPCR machine
Use input RNA samples normalized to a concentration of 1 ng/μl RNA.
Thaw frozen qPCR kit components on ice. Mix and centrifuge briefly.
Swirl mastermix bottle gently to mix.
Calculate number of samples, including the no-template control.
Set up duplicate PCR reactions for each template/assay combination, using the "attached spreadsheet":http://www.nature.com/protocolexchange/system/uploads/4033/original/Master_Mixes.xlsx?1447058311 to scale the following per reaction mixture:
See figure in Figures section.2.14 Mix and centrifuge briefly.
2.15 Distribute into 0.2 ml qPCR wells, 10 μl per well.
2.16 Seal, mix, and centrifuge briefly.
2.17 Place in qPCR machine and run the following program:
See figure in Figures section.The authors declare no competing financial interests.
Supplementary Document 1 Master Mixes
This protocol has been posted on Protocol Exchange, an open repository of community-contributed protocols sponsored by Nature Portfolio. These protocols are posted directly on the Protocol Exchange by authors and are made freely available to the scientific community for use and comment.
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