Transcript first strand cdna synthesis supermix kit

We used two datasets (DFKZ 2018 and GSE70770), as described in the “Data preparation” section, to validate the RS model. We transformed the data into the TPM format to ensure consistency with the training dataset. We applied the RS model to the validation datasets and divided them into two groups according to their respective medians. We then performed corresponding survival and ROC analyses for comparison with the results of the training cohort.
We also constructed a violin plot to visualize the expression of the RS component genes in normal prostate/PCa tissues from the UCSC data. We detected the expression of these genes in 15 pairs of PCa and matched adjacent normal prostate tissues by performing quantitative real-time polymerase chain reaction (qRT–PCR) analysis (Additional file 4).
According to the product protocol, total RNA was extracted using TRIzol (Invitrogen, Waltham, MA, USA), and cDNA was synthesized via reverse transcription using the Transcript First‐Strand cDNA Synthesis Supermix Kit (Transgen Biotech, Beijing, China). SYBR premix Ex Taq II (Takara, Dalian, China) was used to detect the relative expression of the genes included in the model using qRT–PCR, and GAPDH was used as the internal reference. All reactions were repeated three times. Relative expression levels of these genes were calculated using the 2^−ΔΔCT method.

The GMECs were screened with 500 mg/ml G418 (Sigma). A TranScript First-Strand cDNA Synthesis SuperMix Kit (Transgen) was used for reverse-transcription PCR analysis on total RNA samples. The primers BcF/BcR and hcF/hcR which were specific for BLG and hLA partial cDNAs were shown in S1 Table. Supernatants from the induced mammary epithelial cells were collected every 12 hours. The recovered supernatants were then vacuum freeze-dried and subjected to western blot analysis. The primary rabbit anti hLA antibody (1:1,000) used to detect hLA was from Santa Cruz Biotechnology.

One microgram of RNA from each of the six croup skin samples was used to synthesize first-strand cDNA with the Transcript First-Strand cDNA Synthesis SuperMix Kit (TransGen, Beijing, China). Quantitative RT-PCR was performed in a Roche LightCycler 480 II Real-Time PCR Detection System (Roche, Swiss) device by using the Takara SYBR Premix Ex Taq II Kit (TAKARA, Dalian, China). The β-actin locus was used as the reference gene, and relative expression was calculated with the 2^−ΔΔCT method^{99 (link)}. The primer sequences used for qRT-PCR analysis of TBX3 expression were as follows: forward primer 5′-GAGGCCAAAGAACTTTGGGAT-3′ and reverse primer 5′-GGCATTTCAGGATCTGCCTTA-3′.