Biomark hd

Proteins were measured using the Olink^® Mouse Exploratory Panel* (Olink Proteomics AB, Uppsala, Sweden) according to the manufacturer’s instructions as described previously [17 (link)]. The proximity extension assay (PEA) technology used for the Olink protocol has been well described [18 (link)] and allows 92 analytes to be analyzed simultaneously. Briefly, pairs of oligonucleotide-labeled antibody probes bind to their targeted protein, and if the 2 probes are brought in close proximity, the oligonucleotides will hybridize in a pairwise manner. The addition of DNA polymerase leads to a proximity-dependent DNA polymerization event, which generates a unique PCR target sequence. The resulting DNA sequence was subsequently detected and quantified using a microfluidic real-time PCR instrument (Biomark HD, Fluidigm, München, Germany) (Figure 6A). The data are then quality controlled and normalized using an internal extension control and an interplate control to adjust for intra- and interrun variations. The final assay read-out is presented in Normalized Protein eXpression (NPX) values, arbitrary units on a log2-scale in which a high value corresponds to high protein expression. All assay validation data (detection limits, intra- and interassay precision data, etc.) are available on the manufacturer’s website (www.olink.com; accessed date 1 May 2021).

Primers were designed for 68 genes (4 housekeeping genes and 64 pluripotency or germ layer markers) using an in-house algorithm that targeted: (1) a melting temperature of 60°C; (2) regions that spanned multiple exons when possible to minimize genomic DNA signal contamination; and (3) exons that are shared between multiple isoforms. In addition, the primers were tested and showed a single dominant melting curve peak consistent with a dominant amplification product (see Table S2 for a list of primers).
To select the most consistently expressed housekeeping gene for normalization, we tested four housekeeping genes (RPS29, GAPDH, RPL22, and DSG2). We selected RPS29 because this gene had the least variable Ct distribution across all samples (SD = 0.827), whereas the other three housekeeping genes had higher variability (SD = 1.629, 1.623, and 1.642, respectively). The exclusion of these three housekeeping genes resulted in the expression of 65 genes being utilized (1 housekeeping and 64 marker genes).
RNA was isolated from samples using either TRIzol, Qiagen RNeasy Mini Kit, or Qiagen AllPrep DNA/RNA kit. cDNA was produced using Superscript III from 100–500 ng input RNA using oligo(dT)20. Samples were prepared following the Fluidigm Advanced Development Protocol v37, and qPCR was performed on the Fluidigm BioMark HD using EvaGreen and the GE96x96 Fast PCR protocol.

Genes identified in the discovery phase were considered for validation by RT-qPCR, in addition to a reference gene—Glyceraldehyde 3-phosphate dehydrogenase (GAPDH)—which was used to normalize the RNA input of each sample. Primers were designed as described in Supplementary Table 2. The validation was performed using the Biomark HD (Fluidigm) and the 192.24 Dynamic Array integrated fluidic circuit (IFC) following manufacturer instructions. The IFC required the following 3 steps: reverse transcription, pre-amplification, and RT-qPCR (Supplementary Methods).

High-throughput gene-expression analysis with EvaGreen dye on a BioMark HD real-time PCR system was used to measure gene expression according to manufacturer’s recommendations (Fluidigm, San Francisco, CA, USA). All reagents were purchased from Fluidigm unless otherwise indicated. Briefly, cDNA was synthesized from 2 µg of total RNA using Superscript II reverse transcriptase and oligo-dT (Invitrogen) with specific target amplification and exonuclease I (New England Biolabs) treatment. qPCR reactions were performed using a 96 × 96 dynamic array and integrated fluidic circuit. Each sample inlet was loaded with 2.5 µL of 2 × SsoFast EvaGreen supermix with low ROX (Biorad), 0.25 µL of 20×DNA-binding dye sample loading reagent, and 2.25 µL of specific target amplification and exonuclease-I-treated sample. Assays were performed for mitochondrial-coded complex I subunits ND1, ND2, ND3, and ND4, complex III subunit CYB, complex IV subunit COX1, ATP synthase subunit ATP6, and nuclear-encoded complex I subunits NDUFA3, NDUFA11, NDUFA13, NDUFB8, NDUFS8, and NDUFV1, complex IV subunit COX7A2, ATP synthase subunits ATP5G1 and ATP5L, and master regulator PGC-1α. All measurements were performed in duplicate. Primer sequence information is available in Supplementary Table 4. Reference genes ACTB, RPL32, and RPS11 were used to normalize expression values.