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 method⁶. This approach is appropriate for investigating physiological changes in target gene expression levels. However, relative quantification of circulating miRNAs has limited utility because of their small quantities. In addition, technical variation makes it difficult to compare the results from different studies, because different laboratories customize the RT-qPCR experimental protocols differently, which leads to inconsistent or even contradictory results from different studies⁷.

In view of the concerns mentioned above, absolute quantification might be more suitable for the assessment of the small quantities of miRNAs in body fluids. The absolute quantification method uses a standard curve generated from known concentrations of synthetic RNA oligonucleotides that are identical in sequence to the corresponding target miRNA⁸. The Health and Environmental Sciences Institute (HESI) Technical Committee on Genomics recently conducted comprehensive studies to compare the results of absolute measurements of plasma miRNAs, across multiple test sites. The results showed that using a standard protocol for the absolute quantitation of miRNAs yielded comparable results across the multiple test sites⁹. The RT-qPCR assay method described in the present study is almost identical to the HESI's standard protocol, which includes multiplexed analysis of multiple miRNA targets, and pre-amplification to aid the detection of low expression miRNAs.

In this study, a fixed volume (200 µL) of EDTA-plasma prepared from the blood collected from the femoral vein of conscious cynomolgus monkeys (n = 50) was used¹⁰. The following protocol describes the procedure for the preparation of plasma samples, extraction of miRNA, and RT-qPCR, including pre-amplification. More importantly, additional technical information about the protocol has been included, so that the quantity of target miRNAs in the samples can be validated in combination with a well-qualified process. First, the standard curve of each miRNA was validated for its individual detection range, prior to its quantification in biological samples. Second, the quality of the current methodology was comprehensively evaluated by means of Cq values of an external control (cel-miR-238). Therefore, this platform yields more informative and reliable data for comparing results from different studies or laboratories.

The profiles of 8 miRNAs have been included in this report as representative results from the assay method described here. These miRNAs have been proposed as potential safety biomarkers associated with tissue injury to the liver (miR-122 and miR-192), heart (miR-1, miR-208a, miR-208b, and miR-499a), and skeletal muscle (miR-133a and miR-206) in rodents and humans^3,11,12,13.

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 into a 2 mL microtube, followed by centrifugation at 16,000 x g at 4 °C for 5 min to remove cell debris and residual platelets.
   NOTE: Quantification of miRNAs can be affected significantly by platelet contamination¹⁶.
5. Place 200 µL aliquots of the supernatant into fresh 2 mL-microtubes, and store at -80 °C until use.
   NOTE: A fixed volume of each sample is used for RNA extraction; therefore, the volume of the aliquot must be exact.

2. RNA Extraction

1. Thaw frozen samples on ice (from step 1.5).
   1. Keep sample cold on ice during RNA extraction. Chill lysis reagent and chloroform on ice prior to use.
2. Add 5 volumes (1000 µL) of lysis reagent, containing monophasic solution of phenol and guanidine isothiocyanate, to the sample (200 µL), and mix vigorously by vortexing for 1 min.
3. Add 5 µL of 5 nM synthetic Caenorhabditis elegans miRNA (Syn-cel-miR-238-3p).
4. Add 1 volume (200 µL) of chloroform, and mix vigorously by vortexing for 1 min.
5. Keep on ice for 2 to 3 min, and then centrifuge the samples at 12,000 x g at 4 °C for 15 min.
6. Transfer the aqueous phase carefully to a new microtube.
   NOTE: Do not transfer any of the organic phase (red) or interphase (white). The volume of the aqueous phase collected should be uniform to avoid handling inconsistency, which results in increased technical variation. The usual amount of aqueous phase transferred in this protocol was 650 µL.
7. Add 1.5 volumes (975 µL) of ethanol, and mix well by pipetting up and down.
8. Transfer the sample into a corresponding column and adapter, followed by vacuum drying for 3 min using vacuum manifolds. If the sample volume is more than 700 µL, repeat this step to process the remaining solution.
   NOTE: Column-based RNA isolation is compatible with vacuum and centrifugation method.
9. Add 200 µL of ethanol to the column, followed by vacuum drying for 1 min.
10. Add 800 µL of RWT Buffer to the column, followed by vacuum drying for 2 min.
11. Add 800 µL of RPE Buffer to the column, followed by vacuum drying for 2 min.
12. Repeat step 2.11.
13. Add 300 µL of ethanol to the column, followed by vacuum drying for 1 min.
14. Place the column onto a new microtube, and centrifuge at 12,000 x g at room temperature (15 to 25 °C) for 1 min.
15. Transfer the column to a new microtube, and add 50 µL of nuclease-free water.
16. Stand at room temperature (15 to 25 °C) for 3 min, and centrifuge at 8,000 × g at room temperature (15 to 25 °C) for 1 min.
17. Re-apply the eluate to the column.
18. Repeat step 2.16, and store eluate at -80 °C until use.

3. cDNA Synthesis

1. Thaw frozen samples (from step 2.18).
2. Prepare known concentration of synthetic RNA oligonucleotides that correspond to target miRNAs.
   1. Use synthetic RNA oligonucleotide for generating a standard curve in qPCR. Stock solution of 1 x 10⁸ copy/µL concentration is prepared for storage purposes.
   2. Dilute the stock solution 10-fold to obtain 1 x 10⁷ copy/µL (Not pre-amplified samples) or 1 x 10⁵ copy/µL (pre-amplified samples) working solution as the highest concentration for constructing the standard curve. In general, a suggested range for the standard curve concentrations is 1 x 10⁷ to 1 x 10² copy/µL (Not pre-amplified samples) or 1 x 10⁵ to 1 x 10⁰ copy/µL (pre-amplified samples).
3. Prepare multiplex RT primer pool by mixing equal volumes of 20x RT primers for target miRNAs.
   NOTE: As shown in Figure 2, a pool containing up to 4 target miRNAs can be made by mixing the 20x RT primers of each of the miRNAs in the pool. Cel-miR-238 (external control) must be included as one of the target miRNAs in each tube. In case of fewer than 4 target miRNAs, add equal volumes of 1/10 TE buffer instead of 20x RT primer.
4. Prepare RT reaction mix: 3 µL of RT primer pool (from step 3.3), 0.15 µL of 100 mM dNTPs with dTTP, 1 µL of reverse transcriptase (50 U/µL), 1.5 µL 10x RT buffer, 0.19 µL of RNase inhibitor (20 U/µL), and 4.16 µL of nuclease-free water.
5. Mix 10 µL of RT reaction mix with 5 µL of RNA sample (from step 3.1) or oligonucleotides (from step 3.2) by pipetting, and incubate on ice for 5 min.
6. Run reverse transcription on a thermal cycler apparatus: 16 °C for 30 min, 42 °C for 30 min, followed by a final reverse transcriptase inactivation step at 85 °C for 5 min. Store reverse transcribed samples at -80 °C until use.

4. Preamplification (Optional)

NOTE: The miRNAs that show Cq values above 35 or more in subsequent qPCR are pre-amplified.

1. Thaw frozen samples (from step 3.6).
2. Make 10-fold serial dilutions of RT product from synthetic RNA oligonucleotides; 1 x 10⁵ to 1 x 10⁰ copy/µL for generating standard curve.
3. Prepare multiplex assay primer pool by mixing equal volumes (5 µL) of 20x assay primers for target miRNAs with the final concentration of each assay primer being diluted 200-fold by TE buffer in a final volume of 1,000 µL.
   NOTE: The assay primers whose target miRNAs can be detectable in subsequent qPCR without pre-amplification, and those for cel-miR-238 (external control) are not included in primer pool.
4. Prepare pre-amplification reaction mix: 12.5 µL of 2x ready-to-use preamplification reagent, 3.75 µL of assay primer pool (from step 4.3), and 6.25 µL of nuclease-free water.
5. Mix 22.5 µL of pre-amplification reaction mix with 2.5 µL of reverse transcribed sample (from step 4.1) or oligonucleotides (from step 4.2) by pipetting, and incubate on ice for 5 min.
6. Run reaction on a thermal cycler apparatus: 95 °C for 10 min; followed by 12 cycles of 95 °C for 15 s and 60 °C for 4 min. Store pre-amplified samples at -80 °C until use.

5. Quantitative Real-time PCR (qPCR)

1. Thaw frozen samples (from step 3.6 or step 4.6).
2. Dilute the samples 5-fold with sterile water.
3. Prepare 10-fold serial dilutions of the RT product derived from synthetic RNA oligonucleotides; 1 x 10⁷ to 1 x 10² copy/µL (Not pre-amplified samples) or 1 x 10⁵ to 1 x 10⁰ copy/µL (pre-amplified samples) for generating standard curves.
4. Prepare qPCR reaction mix: 10 µL of 2x ready-to-use amplification reagent, 1 µL of 20x assay reagent, containing forward/reverse PCR primer and probe corresponding to target miRNA, and 7 µL of nuclease-free water.
5. Transfer 18 µL from the qPCR reaction mix to the fast optical 96-well reaction plates, and add 2 µL of the diluted samples (from step 5.2 or step 5.3) into the wells.
   NOTE: Samples and standards for qPCR are set up in duplicates.
6. Seal the plate with adhesive film, and centrifuge briefly.
7. Run reaction on a real-time thermal cycler: 95 °C for 20 s, followed by 45 cycles of 95 °C for 1 s and 60 °C for 20 s.

6. Data Analysis

1. Compute the raw copy number of each sample using data analysis software that works with corresponding real-time thermal cycler.
   NOTE: Threshold line is set manually to "1.0" in all plates in the study, to confirm the reproducibility by comparison with their Cq vales. Cut-off level is set at Cq >40 cycles.
2. Calculate the average of the raw copy number of each sample from duplicates.
3. Calculate the correction factor by dividing a copy number of cel-miR-238 in each sample by the average of copy number of cel-miR-238 from all samples in a corresponding tube.
   NOTE: As shown in Figure 2, each tube contains up to 4 target miRNAs including cel-miR-238 (external control). Therefore, correction factor is calculated for each tube using corresponding cel-miR-238 value.
4. Calculate the adjusted copy number by dividing the averaged raw copy number of each sample (from Step 6.2) by the correction factor (from Step 6.3) for each sample.
5. Calculate the absolute copy number by multiplying the adjusted raw copy number of each sample (from Step 6.4) by the the dilution factor for each sample.
   NOTE: The dilution factor is "5" in this protocol, which is derived from Step 5.2.
6. Calculate the efficiencies of PCR amplification from the slope of the plot of the Cq values for each serial dilution against log cDNA concentration.
   NOTE: Formula for calculating the efficiency; E = (10^(-1/slope) -1) x 100 %.
7. Calculate the technical variation using the Cq values of cel-miR-238 from all samples using the formula E = (2 x amplification efficacy)^{ΔCt(Max(Cq)-Min(Cq))}.
   NOTE: Steps 6.5 and 6.7 are used for quality assessment of the procedure.

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