The native and processed sperm sample was centrifuged for 1 min at 3.500 rpm. RNA extraction was performed from 200 μL supernate with the use of the EZ1 Virus Mini Kit v2 (Qiagen) following the manufacturer’s instructions; 60 μL was eluted from the 200 μL starting material, and 5 μL of the eluate was tested by means of reverse-transcription polymerase chain reaction (RT-PCR) with the use of the TaqMan technique. A 113-base-pair amplicon in the E-gene of SARS-CoV-2 was amplified and detected, as previously described with minor modifications (11 ). RT-PCR was performed with the use of an ABI 7500 FAST sequence detector system (PE Applied Biosystems). The thermal protocol described was shortened to 40 cycles of 95°C. We used the LightMix Modular SARS and Wuhan CoV E-gene (cat. no. 53-0776-96) and the LightMix Modular EAV RNA Extraction Control. Moreover, we used the AgPath-ID One-Step RT-PCR Kit (Applied Biosystems cat. no. 4387391; DNA-standard plasmid pEX-A128-nCoV2019-E-gene).
Abi 7500 fast sequence detector system
Manufactured by Thermo Fisher Scientific
Sourced in Germany, United States
The ABI 7500 FAST sequence detector system is a real-time PCR instrument designed for high-throughput nucleic acid detection and quantification. It features a 96-well block format and supports fast cycling protocols. The system utilizes fluorescence-based detection to monitor the amplification of target sequences in real-time.
Automatically generated - may contain errors
Lab products found in correlation
Ez1 virus mini kit v2, by Qiagen (1 mentions)
Agpath id one step rt pcr kit, by Thermo Fisher Scientific (1 mentions)
Lightcycler 1, by Roche (1 mentions)
Qpcr mastermix, by Primerdesign (1 mentions)
Sybr premix ex taq 2, by Takara Bio (1 mentions)
Rt reagent kit, by Takara Bio (1 mentions)
Trizol, by Thermo Fisher Scientific (1 mentions)
3 protocols using abi 7500 fast sequence detector system
1
SARS-CoV-2 RNA Detection by RT-PCR
2
SARS-CoV-2 Detection by qRT-PCR
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For further analysis, 5 μL of the eluate was subjected to qRT-PCR using the real-time TaqMan®-technique where a 113 base pair amplicon in the E-gene of SARS-Cov-2 was generated and detected according to [41 (link)], with minor modifications, as described by Ramani et al. (2020) [24 (link)]. We used the LightMix® Modular SARS and Wuhan CoV E-gene (Cat.-No. 53-0776-96) and the LightMix® Modular EAV RNA Extraction Control and the AgPath-ID® One-Step RT–PCR Kit (Applied Biosystems, Waltham, MA, USA, Cat. No. 4387391). PCR was performed on the ABI 7500 FAST sequence detector system (PE Applied Biosystems, Weiterstadt, Germany).
Gene expression analysis of GAPDH was performed to confirm the presence of a sufficient amount of cells in the samples as suggested in [42 (link)]. To this end, quantitative RT–PCR analysis was performed by using qPCR MasterMix (PrimerDesign Ltd, Chandler’s Ford, UK) with primers #5163 (5′ CCA CTC CTC CAC CTT TGA 3′) and #5164 (5′ ACC CTG TTG CTG TAG CCA 3′) and fluorescence emission was monitored by LightCycler 1.5 (Roche, Basel, Switzerland).
Gene expression analysis of GAPDH was performed to confirm the presence of a sufficient amount of cells in the samples as suggested in [42 (link)]. To this end, quantitative RT–PCR analysis was performed by using qPCR MasterMix (PrimerDesign Ltd, Chandler’s Ford, UK) with primers #5163 (5′ CCA CTC CTC CAC CTT TGA 3′) and #5164 (5′ ACC CTG TTG CTG TAG CCA 3′) and fluorescence emission was monitored by LightCycler 1.5 (Roche, Basel, Switzerland).
3
Quantification of Adipose Tissue mRNA
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Total RNA either from white adipose tissue or brown adipose tissue was isolated using TRIzol (Invitrogen) and reverse transcribed into cDNA by an RT reagent kit (Takara, RR047A) according to the manufacturer’s instructions. Quantitative real-time PCR was conducted using SYBR Premix Ex TaqⅡ (Takara, RR8320A) on an ABI 7500 Fast sequence detector system (Applied Biosystems, Foster City, CA, USA). The relative mRNA expression levels were determined using the 2−ΔΔCT method as described previously. The sequences of primers used in our study were showed in supplemental Table 1.
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