Qpcr platform

This study was reviewed and approved by the ethics committee of the Seventh Affiliated Hospital of Sun Yat-sen University and was adhered to the tenets of the Declaration of Helsinki. All participants provided written informed consent before they were included in the study. Blood samples were collected from 10 patients with SCI, 3 patients with closed noncentral nervous system trauma, and 3 healthy individuals. According to a method described in the literature, ACK Lysing Buffer (Thermo Fisher Scientific, Waltham, MA, USA) was used to lyse red blood cells to obtain white blood cells in blood (33 (link)). The RNA of these white cells was extracted using an RNA extraction kit (Beyotime, Shanghai, China). The RNA was reverse‐transcribed to cDNA using PrimeScript RT Master Mix (TAKARA, Dalian, China), and expression levels of the biomarkers of SCI and different ASI grades were verified by qPCR. PowerUp SYBR reagents (Thermo Fisher Scientific, Waltham, MA, USA) and the qPCR platform from Bio‐Rad (Hercules, CA, USA) were used. The primer sequences used for qPCR were listed in Table 1.

We constructed a similar mouse SCI model according to GSE5296 by using Allen's method. There were three mice in the blank group and the injury group at each timepoint. All animal operations were performed under general anesthesia after inhalation with isopentane (RWD Life Science, Shenzhen, China). Then spinal cord tissues were collected from the injured areas, and RNA was extracted from these tissues using an RNAeasy animal RNA extraction kit (Beyotime, Shanghai, China). The RNA was reverse‐transcribed into DNA using PrimeScript RT Master Mix (TAKARA, Dalian, China). Then the expression levels of hub genes at various timepoints after SCI were verified through qPCR. PowerUp SYBR reagents (Thermo Fisher Scientific, Waltham, MA, USA) and a qPCR platform (Bio‐Rad, Hercules, CA, USA) were used. The primer sequences used for qPCR were listed in Table 1.

We used off-the-shelf primers designed by Applied BioSystems (Life Technologies) for the analysis. The primers are usually designed to span exon–exon junction to target multiple splice variants of one transcript and to target only and specifically the gene of interest, avoiding amplification of genomic DNA. S3 Table lists all genes of interest, the inventoried TaqMan assay IDs (Applied Biosystems), and further relevant information when the manufacturer does not provide primer and probe sequences. To evaluate qPCR assay performance, calibration (standard) curves were generated by performing qPCR on a serial dilution of a prepared template. Each of these dilutions was dispensed into two subarrays of OpenArray plate, leading to six technical qPCR replicates for each single cell sample. To minimize the effect of sampling errors on quantification precision, only sample/assay combinations with at least three quantifiable replicates were considered for preparing the standard curves. The GAPDH assay was not preimmobilized on OpenArray plate but was independently tested on BioRad qPCR platform.