Validation of droplet digital PCR for cytokeratin 19 mRNA detection in canine peripheral blood and mammary gland

Sample collections

The samples were canine peripheral blood (n = 3) and non-neoplastic mammary tissues (n = 3) obtained from the dogs that were presented to the Division of Obstetrics, Gynecology and Reproduction, Small Animal Teaching Hospital, Faculty of Veterinary Science, Chulalongkorn University between January and March 2021. The peripheral blood (5 mL) was collected from clinically healthy dogs based on screening tests including physical examination, hydration status assessment, CBC, serum biochemistry (alanine aminotransferase and creatinine) and diagnostic imaging (thoracic radiography and abdominal ultrasonography). The non-neoplastic mammary tissues (50 mg) were collected from non-tumor mammary glands. The tissue was histopathologically examined to assure that there were no evidences of cancerous cells. Blood and tissue samples were stored immediately at 4°C. Thereafter, the process of RNA extraction was performed within 30–60 min. Synthetic plasmid DNA containing CK19 and HPRT was used as a positive template (Invitrogen, Massachusetts, United States) (see Supplementary Fig. S1).

RNA extraction

Blood of a healthy female dog (5 mL) was added in the ratio of 20 mL of 1X RBC lysis buffer (Cell Signaling Technology, Danvers, Morocco) to 1 mL of blood in a 50 mL centrifuge tube, then centrifuged at 10,000 × g for 5 min at 4°C, and the supernatant was then removed without disturbing the cell pellet. Meanwhile, 50 mg of non-neoplastic canine mammary tissue was lysed and homogenized with tissue homogenizer (FastPrep-24 5G, MP biomedicals, California, United States). The mRNA from blood sample (cell pellet) and homogenized tissue were extracted using TRIzol reagent (ThermoFisher Scientific, Massachusetts, United States), following the protocol described in the kit description. Finally, the RNA pellets were resuspended in 30 µL of RNase-free water and incubated in a heat block set at 55 °C for 10 min. The RNA was stored at −80°C until used. The RNA concentration was determined by a spectrophotometer (NanoDrop ND-1000; Peqlab, Erlangen, Germany) reading at 260 and 280 nm. The samples with a ratio of 1.8–2.0 (260/280) were included in the study. The RNA integrity was measured by a bioanalyzer (RNA600 Nano Chip) (Bioanalyzer2100, Agilent, California, United States). The RNA integrity number was above 520. DNA contamination was then treated with DNase I (TURBO DNase, Invitrogen, Massachusetts, United States), including 200 ng of total nucleic acid solution, 1 × buffer (10 × TURBO DNase Buffer) in the RNA sample, 2 U of DNase I and DEPC-treated water to 50 µL final volume. The reaction was incubated at 37°C for 30 min, and then EDTA was added to a final concentration of 15 mM over 10 min at 75°C to stop the reaction.

cDNA synthesis

Total RNA (1 µg) was reverse transcribed using a cDNA synthesis Kit (iScript Reverse Transcription Supermix Kit, California, United States). The reaction contained 4 µL of iScript RT Supermix, 1 µg of RNA template and nuclease-free water, used to dilute the total volume to 20 µL. The reactions were incubated at 25°C for 5 min, 46°C for 20 min and 95°C for 1 min. No reverse transcriptase (no-RT) controls were also performed as described in the kit. The success of the cDNA synthesis was confirmed by PCR amplification of the canine housekeeping gene (HPRT). The cDNA was stored at −80°C until used.

Primer and probe design

This section describes the design of the primers and probes for CK19 and HPRT, using the Canis lupus familiaris keratin 19 (CK19) mRNA (NM_001253742.1) and theCanis familiaris HPRT mRNA (AY283372.1), respectively. All primers and probes were designed by Primer Express Software Version 3.0.1 (ThermoFisher Scientific, Massachusetts, United States) with default settings, which follow the primer and probe design criteria21. The primers and hydrolysis probes for CK19 and HPRT were synthesized and purified by Macrogen Inc. (Seoul, Korea) (Table 1) and the locations of both primers are shown in Supplementary Figs. S2 and S3 (genome reference: CanFam3.1). To check the specificity of the primers and probes, the Primer-BLAST22 and BLAST23 were performed.

Table 1 Sequences of design primers and hydrolysis probes.

Quantitative real-time PCR reaction

Quantitative real-time PCR (qPCR) method was used first to test the assays before moving to the ddPCR platform. The synthetic plasmid DNA templates were used to test the assay and PCR efficacy. The concentration of this plasmid consists of 100–105 copies of plasmid DNA templates. To test the assay with RNA from one peripheral blood sample and one non-neoplastic mammary tissue, a ten-fold serial dilution of the cDNA from blood and non-neoplastic mammary tissue was made up with nuclease-free water, creating a solution range of no dilution to 1:1000.

The 20 µL qPCR mixture consisted of 10 µL Luna Universal qPCR Master Mix (BioRad, California, United States), 2.0 µL of DNA template, 300 nmol/L forward and reverse primers and 200 nmol/L probes. Three replicates of PCR were run to end point using a thermal cycler (CFX Connect Real-time PCR Detection System, BioRad, California, United States). The setting for PCR reaction was 1 cycle of 95°C for 10 min, 45 cycles of 95°C for 15 s and 45 cycles of 60°C for 30 s. No template control (NTC) was also included.

Droplet digital PCR reaction and the optimization of annealing temperature (Thas)

The PCR protocols followed the MIQE guideline24 (see Supplementary Table S1). For optimization of Thas103 copies of the synthetic plasmid DNA were used as the template, and the gradient temperature between 55 and 65 °C was determined. HindIII (5 U) (New England Biolabs, Massachusetts, United States) was added into the ddPCR reaction to digest a recognition site between the target sequences. There were 2 types of ddPCR assays, including duplex and singleplex. In the duplex assay, both CK19 and HPRT assays were added into the PCR reaction, while in the singleplex ddPCR only CK19 assay or HPRT assay was included in the PCR mixture.

The 20 µL of ddPCR mixture consisted of 10 µL 2 × ddPCR Supermix for Probes (BioRad, California, United States), 2.5 µL of the template DNA samples, 300 nmol/L of forward and reverse primers and 250 nmol/L probes. NTCs also were performed. The ddPCR mixture was loaded into ddPCR 96-well Plates (BioRad, California, United States), mixed and centrifuged for 30 s at 1000 rpm. The plate was placed into the Automated Droplet Generator (AutoDG, BioRad, California, United States) to partition the sample into droplets, which combined oil (Automated Droplet Generation Oil for Probes, BioRad, California, United States) and sample. Next, a 96-well PCR plate containing droplets was sealed with foil by a PX PCR Plate Sealer (BioRad, California, United States). PCR was run to end point using a thermal cycler (T100 Thermal Cycler, BioRad, California, United States). The PCR cycle of ddPCR was performed at 95°C for 10 min, 40 cycles of 94°C for 30 s, 1 min at optimal temperature for annealing and extension, and 1 cycle of 98°C for 10 min.

The droplets were read by a droplet reader (QX200 Droplet Reader, BioRad, California, United States). The droplet reader read each droplet to qualify droplets with a target or without. Data was analyzed with QuantaSoft Software v.1.7.4.0917 (BioRad, California, United States). The quantification measurements of the target molecule were presented as the copies/µL of sample.

Limit of detection (LOD)

The LOD is defined as the lowest PCR copy number concentration that can be discriminated from zero with a level of confidence of 95%, where at least 95% of the replicates are positive, and a minimum of ten replicates should be performed25. To establish the measurement range, the synthetic plasmid DNA was prepared in the concentration range of 50,000–5 copies/µL in three replicates and used as the templates in ddPCR reactions. The copy number of the plasmid DNA were converted from ng obtained from a spectrophotometer (NanoDrop ND-1000; Peqlab, Erlangen, Germany). Then, to determine the LOD, the lowest concentration of the synthetic plasmid was measured by ddPCR with ten replicates.

CK19 detection in peripheral blood and mammary tissue by ddPCR

The cDNA from canine peripheral blood (n = 3) and non-neoplastic mammary tissues (n = 3) was performed under the aforementioned ddPCR conditions for detection of CK19 and HPRT. To verify the method, 50 mg of canine malignant mammary tumor tissue (Tubulopapillary carcinoma) was performed a twofold weight reduction (50–1.56 mg) and then spiked into 5 mL of blood from a healthy female dog as positive controls. All samples were done in three replicates for the ddPCR method.

Statistical analysis

Descriptive statistics were used to describe the copy numbers of CK19 and HPRT. The copy numbers derived from replicates were presented as means and standard deviations.

Ethical approval

All procedures involving the handling and collection of samples were approved by the Institutional Animal Care and Use Committee of Chulalongkorn University (CU-IACUC approval number 2131037) and performed in accordance with relevant guidelines and regulations. Informed consent and permissions were obtained from all the dog owners. This study was carried out in compliance with the ARRIVE guidelines.

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