Transstart tip green qpcr supermix kit

The overnight cultures of WT/pSTV28, XM1/pSTV28, and XM1/pCcpxRA were inoculated into 100 mL fresh LB at 1:100 and incubated at 37 °C with shaking The bacteria were collected for total RNA extraction when they reached the exponential phase. The Spin Column Bacteria Total RNA Purification Kit (Sangon) was used to extract total RNA from cells after collecting the cells by centrifugation and resuspending them in RNase-free water in advance. Reverse transcription was performed using the EasyScript One-Step gDNA Removal and cDNA Synthesis SuperMix kit (Transgen), according to the manufacturer’s instructions. RT-qPCR was performed with RT primers following the instructions of the TransStart Tip Green qPCR SuperMix kit (Transgen) on the CFX96 Real-Time System (BioRad). With the 16S cDNA gene as a housekeeping gene, the quantity of the target genes was normalized [32 (link)]. All of the RT-qPCR assays were repeated at least 3 times.

A549 cells were infected with A/PuertoRico/8/1934(H1N1) virus at an MOI of 0.02, and the compounds were added as described in the section for cytopathic effect assay. The supernatant was collected 24 h post-infection. Virion RNA in the supernatant was extracted with viral RNA isolation kit (Macherey-Nagel, Düren, Germany, Cat. No. 740984.50) as per the manufacturer’s protocol and was reverse transcribed using a cDNA synthesis kit (TransGen Biotech, Beijing, China, Cat. No. AT311-02) with reverse transcription (RT) primer (5′-GAATGGACGAAAAACAAGAATTGC-3′). The RT products were then used as templates for qPCR with a TransStart Tip Green qPCR Supermix kit (TransGen Biotech, Beijing, China, Cat. No. AQ142-21); the primer sequences are as follows: Fwd_5′-GAATGGACGAAAAACAAGAATTGC-3′; Rev_5′-CTCAATATGAGTGCAGACCGTGCT-3′. The qPCR assay was performed according to the manufacturer’s instructions on QuantStudio 3 Real-Time PCR system (Applied Biosystems, Waltham, MA, USA). The Ct values were analyzed using the QuantStudio Design and Analysis software (v1.5.1, Applied Biosystems, Waltham, MA, USA). The assay was performed for each compound in three biological replicates and two technical replicates, and the same volume of DMSO (1‰, v/v) was set as the vehicle control. The relative virus titer was determined based on an analysis of Ct values and normalized to vehicle control.

The total cDNA was isolated from 2-hpa oocytes (≥20) using TransScript-Uni Cell to cDNA Synthesis SuperMix for Q-PCR kit (TransGen Biotech, Beijing, China). Then, cDNA was quantified by Q-PCR using a TransStart Tip Green qPCR SuperMix Kit (TransGen Biotech, Beijing, China) on a CFX Connect Real-Time Detection System (Bio-Rad, Hercules, CA, USA) under standard conditions. The cycle threshold (Ct) value used to calculate the relative expression was the average of three replicates and was normalized against that of the reference gene (Gapdh). The primer information is summarized in Table 1. The mRNA expression levels were calculated using the 2^-△△Ct method [47 (link)].
Table 1

Information of primers used in RT-qPCR

Genes	Accession ID	Primer Seq (5′→3′)	Product Length, bp	Tm, °C
Btg4	NM_019493.4	F: TGAACAACCCAAAGAGCGTCTACC	103	55
		R: AACCCACGACCATCTGCCAAATG
Orc2	NM_001025378.2	F: TGATTCATGTCTTACGAAGCCT	107	55
		R: AGAAAGTCCAATGTAGGAAGGG
Eif4e	NM_001313980.1	F: ACTTTTGGGCTCTATACAACCA	82	55
		R: ATCCCGTCCTTAAAAAGTGAGT
Miss	NM_001045483.1	F: GTCCTTTAGGTACACAGGGATC	207	55
		R: GATATGACGCTTCAGGAGTAGG
Doc1r	NM_026373.4	F:ACGGACCTGCTGTCTGTCATAGAG	146	55
		R: TTGCGTTCTGTCTCTGCCAAGC
Ythdf2	NM_145393.4	F:TTGCCTCCACCTCCACCACAG	111	55
		R:CCCATTATGACCGAACCCACTGC
Setd2	NM_001081340.2	F: GGAGGCAGACACGGAGACAGAG	139	55
		R: ATCTGGTGGCTCCTGGCTTCTC
Gapdh	NM_008084.3	F: CATGGCCTTCCGTGTTCCTA	104	55
		R: GCCTGCTTACCACCTTCTT

Total RNA was extracted from S. aureus Newman treated with 0.5× the MIC of crizotinib for 1 and 2 h using the Eastep super RNA extraction kit (Promega, USA) according to the manufacturer’s protocol. The mRNA was reverse transcribed using a TransScript II one-step RT-PCR SuperMix kit (TransGen, China). Real-time quantitative PCR (RT-qPCR) was performed with the TransStart Tip Green qPCR SuperMix kit (TransGen, China). The expression of target genes was assayed using the StepOne system (Applied Biosystems). The fold changes of selected genes were normalized to the threshold cycle (C_T) value of 16S RNA amplified from the corresponding sample and calculated using the 2^−ΔΔCT method (37 (link)). The primer sequences are listed in Table 1. All experiments were performed in three biological replicates.