Transcript all in one first strand cdna synthesis supermix for qpcr kit

The changes in expression of six randomly selected genes were confirmed by conducting qRT-PCR using an ABI 7500 fast Real-Time PCR System (Applied Biosystems, Waltham, MA, USA). Briefly, the total RNAs were isolated as described previously. A TranScript All-in-One First-Strand cDNA Synthesis SuperMix for qPCR Kit (Transgen Biotech, Beijing, China) was employed to synthesize the cDNA. qRT-PCR was performed following the protocol of the TransStart Top Green qPCR SuperMix kit (Transgen Biotech, Beijing, China). The Primer-BLAST online NCBI tool was used to design primers specific for each DEG (Table S1). The L25 gene was used as the internal reference to normalize the relative expression levels. The gene expression level of C0 (before infection of CBH) and G0 (before infection of G28) were set to one, and the 2^−ΔΔCt method was used to calculate the relative expression level of each assessed gene (Livak and Schmittgen, 2001 (link)). Sequencing data were verified by comparing the expression trends of each gene as determined by qRT-PCR and FPKM values. Three biological technical replicates were performed to validate the results of qRT-PCR.

qRT-PCR was employed to verify the veracity of the transcriptome data with twenty randomly selected DEGs. The specific primers of these DEGs were designed by Primer-BLAST of online tool of NCBI. RNA samples were prepared as described in “RNA extraction, library construction and Transcriptome sequencing” section. The cDNAs synthesized using a TranScript All-in-One First-Strand cDNA Synthesis SuperMix for qPCR Kit (Transgen Biotech, Beijing, China). qRT-PCR was performed following the protocol of TransStart Top Green qPCR SuperMix kit (Transgen Biotech, Beijing, China), on the ABI 7500 fast Real-Time PCR System (Applied Biosystems, USA). The housekeeping gene of β-Actin gene was used as the reference to normalize the relative expression levels, with its primer sequences: F: 5′-ATCCTCCGTCTTGACCTTG-3′, and R: 5′-TGTCCGTCAGGCAACTCAT-3′. The qRT-PCR carried out in 20 μL system at the following conditions: one cycle of 94 °C for 30s; 40 cycles of 94 °C for 5 s, 60 °C for 34 s, and one cycle of 60 °C for 60s. Three biological and technical replicates were performed to validate the results in qRT-PCR tests. The relative gene expression level was quantified by the 2^-ΔΔCt method [81 (link)].

RZ solution (Transgen, China) was used to extract total RNA from peripheral blood mononuclear cells (PBMCs) of 106 newly diagnosed AML patients in our hospital. cDNA was synthesized using a TranScript All-in-One First-Strand cDNA Synthesis SuperMix for qPCR kit (Transgen, China), and TranStart Tip Green Qpcr SuperMix (Transgen, China) was used to assay the HOXA5 mRNA levels. The amplification proceeded as follows: 94°C, 30 seconds, 1 cycle; 94°C, 5 seconds, 60°C, 30 seconds, 40 cycles. The primers were listed in Supplementary Table S8. The 2−ΔΔ CT method was used to calculate relative mRNA expression.