Absolute Quantification of Plasma MicroRNA Levels in Cynomolgus Monkeys, Using Quantitative Real-time Reverse Transcription PCR

Summary

This report describes a protocol for measuring the absolute levels of plasma miRNA, using quantitative real-time reverse transcription PCR with or without pre-amplification. This protocol affords better understanding of the quantity of plasma miRNAs and allows qualitative assessment of corresponding data from different studies or laboratories.

Abstract

RT-qPCR is one of the most common methods to assess individual target miRNAs. MiRNAs levels are generally measured relative to a reference sample. This approach is appropriate for examining physiological changes in target gene expression levels. However, absolute quantification using better statistical analysis is preferable for a comprehensive assessment of gene expression levels. Absolute quantification is still not in common use. This report describes a protocol for measuring the absolute levels of plasma miRNA, using RT-qPCR with or without pre-amplification.

A fixed volume (200 µL) of EDTA-plasma was prepared from the blood collected from the femoral vein of conscious cynomolgus monkeys (n = 50). Total RNA was extracted using commercially available system. Plasma miRNAs were quantified by probe-based RT-qPCR assays which contains miRNA-specific forward/reverse PCR primer and probe. Standard curves for absolute quantification were generated using commercially available synthetic RNA oligonucleotides. A synthetic cel-miR-238 was used as an external control for normalization and quality assessment. The miRNAs that showed quantification cycle (Cq) values above 35 were pre-amplified prior to the qPCR step.

Among the 8 miRNAs examined, miR-122, miR-133a, and miR-192 were detectable without pre-amplification, whereas miR-1, miR-206, and miR-499a required pre-amplification because of their low expression levels. MiR-208a and miR-208b were not detectable even after pre-amplification. Sample processing efficiency was evaluated by the Cq values of the spiked cel-miR-238. In this assay method, technical variation was estimated to be less than 3-fold and the lower limit of quantification (LLOQ) was 10² copy/µL, for most of the examined miRNAs.

This protocol provides a better estimate of the quantity of plasma miRNAs, and allows quality assessment of corresponding data from different studies. Considering the low number of miRNAs in body fluids, pre-amplification is useful to enhance detection of poorly expressed miRNAs.

Introduction

An increasing number of studies have been exploring microRNAs (miRNAs) as biomarkers for the diagnosis and prognosis of cancers, or monitoring and detecting other diseases in nonclinical and clinical studies^1,2,3. Quantitative real-time reverse transcription PCR (RT-qPCR) is one of the most common methods used to assess individual target miRNAs, because this technique is more sensitive than microarray⁴ and RNA sequencing based platforms⁵. In general, miRNA expression is measured relative to a reference sample using the ΔCq ....

Protocol

All experiments were approved by the Institutional Animal Care and Use Committee of Daiichi Sankyo Co., Ltd.

1. Sample Preparation

1. Collect blood (at least 0.5 mL) from the femoral vein of cynomolgus monkeys into EDTA 2K-containing tubes.
   NOTE: Citrate and heparin are not acceptable because these anticoagulants inhibit subsequent PCR^14,15.
2. Place the collected samples immediately on ice and process for plasma isolation within 2 hours of collection.
3. Centrifuge the samples at 10,000 x g at 4 °C for 5 min.
4. Transfer the supernatant int....

Results

Workflow of miRNA assay by RT-qPCR and quality assessment
Figure 1 shows the workflow of miRNA assay from blood samples using qPCR¹⁰. The quality of the experiments can be verified by including cel-miR-238 as an external control. This will reveal technical variations in RNA extraction and subsequent RT-qPCR processes. In this study, the mean ± SD of the Cq values computed from 50 samples was 21.0 ± 0.4 (

Discussion

Our comprehensive assessment provided a more rigorous statistical analysis of the extent of the dynamic range, which clearly indicated that the magnitude of variation between individual samples was extremely different among the miRNAs tested. Although these variations may be attributable to their small quantities in body fluids, it should be noted that these data reflect not only biological variations, but also technical variations. Most of the technical variation can be assessed by means of Cq values of the external con.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
BD Microtainer tube (K2EDTA)	Becton, Dickinson and Company	365974	For blood collection
Eppendorf PCR Tubes, 0.2 mL	Eppendorf	0030124359
Eppendorf Safe-Lock micro test tubes  1.5 mL	Eppendorf	0030120086
Eppendorf Safe-Lock micro test tubes  2.0 mL	Eppendorf	0030120094
Synthetic oligonucleotide	Hokkaido System Science	-	Individual miRNA (0.2 μmol,HPLC grade)
Tris-EDTA Buffer  (pH 8.0)	Nippon Gene	314-90021	TE buffer
Buffer RPE	QIAGEN	-	Contents in miRNeasy mini kit
Buffer RWT	QIAGEN	-	Contents in miRNeasy mini kit
miRNeasy Mini Kit	QIAGEN	217004
Nuclease-Free Water	QIAGEN	129114
QIAzol Lysis Reagent	QIAGEN	-	Contents in miRNeasy mini kit
Syn-cel-miR-238-3p miScript miRNA Mimic	QIAGEN	219600	ID:MSY0000293, 5 nmol
SC Adapters	TAIGEN Bioscience Corporation	S0120	For RNA extraction
VacEZor 36 Complete System	TAIGEN Bioscience Corporation	M3610	For RNA extraction
7900HT Fast Real-Time PCR System	Thermo Fisher Scientific Inc.	4351405	Fast 96-Well Block
GeneAmp PCR System 9700	Thermo Fisher Scientific Inc.	9700
MicroAmp Fast Optical 96-Well Reaction Plate	Thermo Fisher Scientific Inc.	4346907
MicroAmp Optical Adhesive Film	Thermo Fisher Scientific Inc.	4311971
TaqMan Fast Advanced Master Mix	Thermo Fisher Scientific Inc.	4444557
TaqMan MicroRNA Assays (cel-miR-238-3p)	Thermo Fisher Scientific Inc.	4427975	Assay ID: 000248
TaqMan MicroRNA Assays (hsa-miR-122-5p)	Thermo Fisher Scientific Inc.	4427975	Assay ID: 002245
TaqMan MicroRNA Assays (hsa-miR-133a-3p)	Thermo Fisher Scientific Inc.	4427975	Assay ID: 002246
TaqMan MicroRNA Assays (hsa-miR-1-3p)	Thermo Fisher Scientific Inc.	4427975	Assay ID: 002222
TaqMan MicroRNA Assays (hsa-miR-192-5p)	Thermo Fisher Scientific Inc.	4427975	Assay ID: 000491
TaqMan MicroRNA Assays (hsa-miR-206)	Thermo Fisher Scientific Inc.	4427975	Assay ID: 000510
TaqMan MicroRNA Assays (hsa-miR-208a-3p)	Thermo Fisher Scientific Inc.	4427975	Assay ID: 000511
TaqMan MicroRNA Assays (hsa-miR-208b-3p)	Thermo Fisher Scientific Inc.	4427975	Assay ID: 002290
TaqMan MicroRNA Assays (hsa-miR-499a-5p)	Thermo Fisher Scientific Inc.	4427975	Assay ID: 001352
TaqMan MicroRNA Reverse Transcription Kit	Thermo Fisher Scientific Inc.	4366597
TaqMan PreAmp Master Mix (2×)	Thermo Fisher Scientific Inc.	4391128
Chloroform	Wako Pure Chemicals	035-02616
Ethanol (99.5)	Wako Pure Chemicals	057-00456