This protocol describes how to perform qRT-PCR on microRNAs and mRNAs isolated from extracellular vesicles or other extracellular particles in order to quantify the amount of extracellular RNA present in the original sample.
Method Article
qRT-PCR
https://doi.org/10.1038/protex.2015.119
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This protocol describes how to perform qRT-PCR on microRNAs and mRNAs isolated from extracellular vesicles or other extracellular particles in order to quantify the amount of extracellular RNA present in the original sample.
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 performing qRT-PCR is one of the final steps in the process, allowing quantification of the amount of extracellular RNA found in a sample or biofluid.
For miRNAs
1.1 Normalize input RNA samples to a concentration of 1 ng/μL.
1.2 Thaw RT kit components on ice. Mix gently and centrifuge briefly.
1.3 Calculate number of RT reactions, including no-input control.
1.4 Make RT master mix on ice, using "attached spreadsheet":http://www.nature.com/protocolexchange/system/uploads/4051/original/Master_Mixes.xlsx?1447065271 to scale the following per reaction mixture:
See figure in Figures section.1.5 Mix and centrifuge briefly.
1.6 Combine in 0.2 ml qPCR well:
1 μL input RNA
11 μL RT master mix
3 μL 5X RT primer
1.7 Seal, mix, and centrifuge briefly.
1.8 Incubate on ice for 5 minutes.
1.9 Place in thermocycler and run the following program:
16°C 30 minutes
42°C 30 minutes
85°C 5 minutes
4°C Hold
The reactions may be stored at -20°C at this point.
1.10 Thaw frozen PCR kit components on ice. Mix and centrifuge briefly.
1.11 Swirl mastermix bottle gently to mix.
1.12 Calculate number of samples, including no-input and no-template controls for each miRNA assay.
1.13 Set up triplicate PCR reactions for each template/assay combination, using the "attached spreadsheet":http://www.nature.com/protocolexchange/system/uploads/4051/original/Master_Mixes.xlsx?1447065271 to scale the following per reaction mixture:
See figure in Figures section.1.14 Mix and centrifuge briefly.
1.15 Distribute into 0.2 ml qPCR wells, 10 μl per well.
1.16 Seal, mix, and centrifuge briefly.
1.17 Place in qPCR machine and run the following program:
1 cycle of: 95°C 10 minutes
40 cycles of: 95°C 15 seconds
60°C 60 seconds
For mRNAs
2.1 Normalize input RNA samples to a concentration of 1 ng/μl.
2.2 Thaw RT kit components on ice. Mix gently and centrifuge briefly.
2.3 Calculate number of RT reactions, including no-input control.
2.4 Make RT master mix on ice, using "attached spreadsheet":http://www.nature.com/protocolexchange/system/uploads/4051/original/Master_Mixes.xlsx?1447065271 to scale the following per reaction mixture:
See figure in Figures section.2.5 Mix and centrifuge briefly.
2.6 Combine in 0.2 ml qPCR well:
1 μL input RNA
9 μL RT master mix.
2.7 Seal, mix, and centrifuge briefly.
2.8 Keep on ice until you are ready to put into thermocycler.
2.9 Place in thermocycler and run the following program:
25°C 10 minutes
37°C 120 minutes
85°C 5 minutes
4°C Hold
The reactions may be stored at -20°C at this point.
2.10 Thaw frozen PCR kit components on ice. Mix and centrifuge briefly.
2.11 Swirl mastermix bottle gently to mix.
2.12 Calculate number of samples, including no-input and no-template controls for each miRNA assay.
2.13 Set up triplicate PCR reactions for each template/assay combination, using the "attached spreadsheet":http://www.nature.com/protocolexchange/system/uploads/4051/original/Master_Mixes.xlsx?1447065271 to scale the following per reaction mixture:
• For the TaqMan Assays, set up this reaction mixture:
See figure in Figures section.• For the Universal RNA Spike I Assay, set up this 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:
1 cycle of: 50°C 2 minutes
95°C 10 minutes
40 cycles of: 95°C 15 seconds
60°C 60 seconds
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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