DNA in E. coli was isolated with reference to the Molecular Cloning (Green and Sambrook 2012 ). Bacterial RNA was extracted by RNAprep Pure Cell/Bacteria Kit (TIANGEN Biotech (Beijing) Co., Ltd., Beijing, China) according to the manufacturer’s instructions. Then, reverse transcription of RNA was conducted to generate complementary DNA (cDNA) using PrimeScript RT reagent Kit with genomic DNA (gDNA) Eraser (Takara Bio Inc., Dalian, China). The abundances of ampR and efflux pump gene (acrA) in DNA and cDNA were quantified by quantitative real-time polymerase chain reaction (qPCR) using SuperReal PreMix (Probe) Kit (TIANGEN Biotech (Beijing) Co., Ltd., Beijing, China) on LightCycler 96 platform (Roche, Basel, Switzerland). Here, multidrug efflux pump AcrAB-TolC encoded gene acrA was chosen as it can pump out a broad range of antibiotics from cell compartment and plays a vital role in the development of antibiotic resistance in bacteria (Nolivos et al. 2019 (link); Poole 2005 (link); Xuan et al. 2017 (link)). The abundance of ARGs was calculated as the ARGs copies divided by the total concentration of DNA for ampR (copies/ng DNA) or RNA for acrA (copies/ng RNA) (Hu et al. 2021 (link); Ruiying et al. 2021 (link)). Detailed descriptions of DNA extraction, RNA reverse transcription, and qPCR test are presented in the Supporting Information (SI) (S1-S4) .
Primescript rt reagent kit with genomic dna
Manufactured by Takara Bio
Sourced in Japan
The PrimeScript RT reagent kit with genomic DNA is a laboratory equipment product designed for reverse transcription of RNA. It includes reagents necessary for the reverse transcription process, as well as the ability to handle genomic DNA contamination.
Automatically generated - may contain errors
Lab products found in correlation
Sybr premix ex taq 2, by Takara Bio (2 mentions)
Trizol reagent, by Thermo Fisher Scientific (2 mentions)
Lightcycler96 platform, by Roche (1 mentions)
Superreal premix probe kit, by Tiangen Biotech (1 mentions)
Rnaprep pure cell bacteria kit, by Tiangen Biotech (1 mentions)
Cfx connect real time system, by Bio-Rad (1 mentions)
Trizol protocol, by Thermo Fisher Scientific (1 mentions)
Abi 7900ht fast real time pcr system, by Thermo Fisher Scientific (1 mentions)
Trizol, by Transgene (1 mentions)
5 protocols using primescript rt reagent kit with genomic dna
1
Quantifying Antibiotic Resistance Genes in E. coli
2
Total RNA Extraction and Real-Time PCR
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Total RNA was extracted using the standard TRIzol protocol (Invitrogen, Carlsbad, CA, USA). Then, 1,000 ng of RNA was used for the reverse transcription to synthesize cDNA with the PrimeScript RT reagent kit with genomic DNA (gDNA) Eraser (TaKaRa, Kusatsu, Japan). SYBR Premix Ex Taq II (TaKaRa, Kusatsu, Japan) was used in the real-time PCR reaction system and then carried out on a Bio-Rad CFX Connect real-time system. The related primers are listed in Table S6 .
3
Quantifying Key Gene Expression in Tissues and Cells
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The mRNA expression of FTO, E2F1, and NELL2 in tissues and cells was determined with qRT-PCR. Total RNA was utilizing the TRIzol reagent (Thermo Fisher Scientific, Waltham, MA, USA), and 1 μg of it was reversely transcribed into cDNA following the instructions of the PrimeScript RT Reagent Kit with genomic DNA (gDNA) Eraser (Takara, Shiga, Japan). Then, the qRT-PCR was performed using SYBR Premix Ex Taq II (Takara, Japan) and ABI PRISM 7500 RT-PCR system (ABI, Foster City, CA, USA), with β-glucuronidase (GUSB) as the internal control. The relative quantification (2−△△CT) was used to calculate the relative transcription level of target genes: △△Ct = △Ct experimental group − △Ct control group, △Ct = Ct (target gene) − Ct (internal control). Each sample was repeated in three wells. The primers (Invitrogen) involved were listed in Table S3 .
4
Quantitative Real-Time PCR of Adipose Tissue
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Total RNA from adipose tissues was extracted using TRIzol (TransGen Biotech, Beijing, China). Complementary DNA was synthesized using the PrimeScript RT reagent kit with genomic DNA (Takara) according to the manufacturer’s protocol. PCR was performed and quantified using SYBR Green real-time PCR Master Mix (TransStart Top Green qPCR SuperMix, TransGen Biotech, Beijing, China) with an ABI 7900HT Fast Real-Time PCR System (Applied Biosystems, Inc.). Data were obtained after PCR cycling; gene expression levels were calculated by the 2-ΔΔCT method and normalized to β-actin measured in parallel. The primer sequences are provided in Table 1 .
5
Transcriptome Analysis of Conceptus and Placenta
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Total RNA was isolated from the conceptuses and the placental tissues using Trizol reagent (Invitrogen, Carlsbad, CA, USA) following the manufacturer’s instructions. The integrity of RNA was determined by electrophoresis in 1% agarose gel. The quantity and purity of RNA were determined using a NanoDrop™ 2000 spectrophotometer (Thermo Scientific). Genomic DNA elimination and cDNA synthesis were conducted using PrimeScript RT Reagent Kit with genomic DNA (gDNA) Eraser (Perfect Real Time, TaKaRa) according to the manual. For each sample, 1 μg of total RNA was used for each 20 μL reverse transcription reaction system. All cDNA samples were preserved at −20 °C.
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