Taqman gene expression primers andprobes

Single cells were sorted by FACS into individual wells of 96 well plates. After
cells lysis, mRNA levels were measured by microfluidic single cell qPCR using
the Biomark system (Fluidigm, CA) according to the manufacturer’s
instructions. This resulted in 48 gene expression values (measured in threshold
cycles, Ct) for each one of the cells sorted. We analyzed approximately 160
cells from fetal human kidney after culturing for a single passage. qPCR
standard curves were created using serial dilutions of
“bulk” RNA containing a mixture of HeLa total RNA and
RNA from adult and fetal human kidneys. TaqMan gene expression primers and
probes were purchased from ThermoFisher Scientific. We clustered over the
following genes: NCAM1 (Assay ID Hs00941830_m1), PROM1 (CD133, Assay ID
Hs01009250_m1), and CDH1 (E-Cadherin, Assay ID Hs01023894_m1).
For clustering analysis, we standardized the expression levels of each gene
individually by subtracting the mean and dividing by 3 times the standard
deviation. Then, all values were truncated into the range [−1, +1].
Clustering was performed using complete linkage and correlation distance
(Matlab).

RNA was isolated from 5 mL of culture with an Invitrogen PureLink™ RNA Mini Kit. To ensure that no residual DNA was present, the Invitrogen™ Ambion™ TURBO DNA-free kit was also used before conversion to cDNA. Concentrations of each cDNA sample were determined using a ThermoFisher Scientific NanoDrop 2000, and 2 µg of template was used in the Applied Biosystems (Foster City, CA, USA) High Capacity RNA-to-cDNA kit.
All qPCR reactions were performed in with three biological and three technical replicates on a PikoReal Real Time PCR System (ThermoFisher Scientific) with TaqMan^® Gene Expression primers and probes (Supplementary Table S3) under the following conditions: 95 °C for 10 min; (95 °C for 1 min, 60 °C for 30 s) for 35 cycles; and 72 °C for 7 min [26 (link)]. Analysis was completed using PikoReal Software 2.2 (ThermoFisher Scientific).
Standard curves were obtained using serially diluted gene targets of known concentrations that were amplified via PCR (Supplementary Table S1). Triplicate samples of each dilution were used as template in qPCR and the recorded Cq was graphed versus DNA concentration. Standards for the gene target were included with all experimental sample plates to ensure consistent readings.

Tissue samples were homogenized in guanidium thiocyanate (Trizol, Carlsbad, CA). RNA was extracted per manufacturer’s recommendation and reverse transcribed with iScript RT Supermix (Biorad, Hercules, CA). RT-PCR was performed on a CFX Connect real time system (Biorad) with Taqman gene expression primers and probes (Applied Biosystems, Foster City, CA). Relative gene expression was analyzed by the ΔCt method (Livak and Schmittgen, 2001 (link)).