Primescript rt polymerase

We obtained 52 breast cancer tissues and normal breast tissues from patients who underwent a breast cancer removal in Union Hospital of Tongji Medical College. The studies involving human participants were reviewed and approved by the Ethics Committee of Union Hospital of Tongji Medical College. The patients/participants provided their written informed consent to participate in this study. Histological diagnosis and tumor grade were assessed by three experienced pathologists in accordance with the 2010 American Joint Committee on Cancer staging system.
The total RNA of breast cancer tissues and normal tissues was extracted with the TRIzol reagent (Vazyme, Nanjing, China). The synthesis of cDNAs corresponding to the mRNAs of interest depended on PrimeScript RT-polymerase (Vazyme) and SYBR-Green Premix (Vazyme) with specific PCR primers (Sangon Biotech Co., Ltd, Shanghai, China). The specific primers used were as follows: GAPDH-F: GGG​AAG​GTG​AAG​GTC​GGA​GT; GAPDH-R: GGG​GTC​ATT​GAT​GGC​AAC​A; BCL2-F: ACT​GGC​TCT​GTC​TGA​GTA​AG; BCL2-R: CCT​GAT​GCT​CTG​GGT​AAC. The fold-changes were calculated with the 2^−ΔΔCt method. The difference in BCL2 expression and the prognosis of BCL2 in breast cancer were evaluated with Student’s t-test and the Kaplan–Meier analysis in GraphPad Prism 7 software (GraphPad, Inc., La Jolla, CA, United States).

Total RNA was extracted using TRIzol reagent (Vazyme, Nanjing, China). Subsequently, cDNAs were synthesized using Prime Script RT-polymerase (Vazyme). SYBR Green Premix (Vazyme) with specific PCR primers (Sangon Biotech, Shanghai, China) were used to detect the expression level of corresponding gene RNA. The primer sequences are provided in Supplementary Table 2. The fold-changes were calculated using the 2^−ΔΔCT method.

Total RNA was extracted from tissues using TRIzol reagent (Vazyme, Nanjing, China). PrimeScript RT-polymerase (Vazyme) was used to reverse-transcribe cDNAs corresponding to the mRNAs of interest. qRT-PCR was performed using SYBR-Green Premix (Vazyme) with specific PCR primers (Sangon Biotech Co., Ltd, Shanghai, China). Glyceraldehyde-3-phosphate dehydrogenase was used as an internal control. The 2^−ΔΔCt method was used to calculate fold-changes. Primer sequences are listed in Additional file 1: Table S1.

Proteins were extracted and western blot was performed as previous described [18 (link)]. Total RNA was extracted using TRIzol reagent (Tiangen, China). cDNAs were reverse transcribed into mRNA through PrimeScript RT-polymerase (Vazyme). qRT-PCR was carried out by using SYBR-Green Premix (Vazyme) with specific PCR primers (ThermoFisher, USA), with Glyceraldehyde-3-phosphate dehydrogenase as an internal reference. The gene expression level was analyzed using the 2^−ΔΔCt method. All steps of qRT-PCR follow the instructions.

TRIzol reagent (Vazyme) was used to isolate total RNA. The synthesis of cDNAs corresponding to the mRNAs of interest was conducted using PrimeScript RT‐polymerase (Vazyme), SYBR‐Green Premix (Vazyme), and specific PCR primers (Sangon). Glyceraldehyde‐3‐phosphate dehydrogenase was used as an internal control. The 2^−ΔΔCt method was used to calculate fold‐changes. The difference in the expression of ACE2 and the prognosis of ACE2 in breast cancer were explored with Student's t test and Kaplan–Meier analysis.