Canine and Murine Cell Titration Standard Curve

In order to screen calvarial defects for the presence of canine cells post-implantation, murine and canine RNA were first used to create species-specific primers for real-time quantitative PCR (qPCR). Murine fibroblasts (C3H10T1/2, ATCC, Manassas, VA) and cMSCs were cultured in CCM. Two preparations of C3H10T1/2 and three preparations of cMSCs (1 × 10⁶ cells/prep) were isolated and used to extract RNA using the High Pure RNA Isolation Kit (Roche Diagnostics, Basel, Switzerland). RNA samples were quantified via nanodrop (Thermofisher, Waltham, MA) to determine RNA yields and were normalized to identical RNA values prior to generation of cDNA (SuperScript III cDNA kit, Invitrogen). For qPCR, cDNA was amplified in a 20 µL reaction containing SYBR Green PCR master mix (Fast SYBR Green, Applied Biosystems, City, State) on a CFX96 Real-Time System (Bio-Rad, City, State). Canine RPL13A and GAPDH (Peters et al., 2007), murine RPL13A and GAPDH made using primer-blast (Ye et al., 2012), were used to create species-specific housekeeping primers (Sigma, St. Louis, MO; Figure 2B). The two murine and three canine cDNA preparations were assessed in duplicate with qPCR using both murine and canine RPL13A and GAPDH (4 primer pairs) to confirm species specificity.

Next, murine and canine RNA were used to determine a correlation of threshold cycles and create a standard curve for minimum and maximum numbers of detectable cells. Murine fibroblasts (C3H10T1/2) or cMSCs (2 × 10⁶) were used to extract RNA using the High Pure RNA isolation kit (Roche Diagnostics). Samples were quantified using the nanodrop to obtain RNA concentrations. Once the RNA concentrations for 2 × 10⁶ cMSCs was determined, cMSC RNA samples were titrated from 2 × 10⁶ cells in serial dilutions down to the equivalent of two cells. Titrated canine RNA samples were placed into 36 or 500 µg murine RNA to represent a low or high background of murine RNA (Figure 3A). Each of the mixed canine and murine RNA samples were used to create cDNA (SuperScript III cDNA kit, Invitrogen). qPCR was performed as described above using canine RPL13Aand cycle threshold data for each cell titration were interpolated to a standard curve in Prism version 8.00 for Mac (GraphPad Software, La Jolla, CA). qPCR amplification of canine RPL13A (Figure 3B) was similar in either the low concentration of background murine RNA (36 μg; red lines) or the high concentration of background murine RNA (500 μg; blue lines). Therefore, the standard curve to interpolate detection of cMSCs at day 10 was based on the cMSC cell titrations mixed with low concentration of murine RNA (Figure 3C).

Generation of a qPCR-based cMSC standard curve to detect cMSCs in calvarial defects at Day 10. (A): Serial dilutions of RNA were performed on RNA extracted from 2 × 10⁶ total cMSCs. RNA samples were added to individual sample tubes containing either 36 or 500 μg of murine RNA to determine if the amount of murine RNA would affect the ability to identify canine RPL13A. cDNA samples were generated and qPCR was performed. (B): qPCR amplification curves are shown for canine RPL13A from cDNA samples generated with either 36 μg (red lines) or 500 μg (blue lines) of murine RNA. (C): Cycle thresholds from Panel A (red lines) were used to generate a standard curve using a linear curve fit technique. RNA samples were isolated from the calvarial defects treated with cMSCs or cMSCs + BMP-2. These samples contained both murine and canine RNA. cDNA samples were generated and qPCR performed for canine RPL13A. Cycle threshold results were interpolated on the standard curve. At Day 10 post implantation, only 2–20 total cMSCs were detected.