We verified four DEGs and four DMGs to study the status of m6A in LD tissues of Queshan Black pigs at different periods. RNA extracted from muscle was reverse-transcribed into cDNA using the Evo M-MLV RT Kit with gDNA Clean for qPCR kit (AG11705, Accurate Biotechnology (Hunan) Co.,Ltd, Changsha, China). q-PCR was performed using the SYBR Green Premix Pro Taq HS qPCR Kit (AG11701, Accurate Biotechnology (Hunan) Co.,Ltd, Changsha, China) using the CFX96 real-time PCR detection system (Thermo Fisher Scientific, USA) according to the instructions. The GAPDH was used as the internal reference gene to normalize the gene expression levels. The relative expression of genes was calculated using the 2−ΔΔCt method. MeRIP-qPCR does not require internal reference genes, and the IP/input ratio is calculated by 2−ΔCt (ΔCt = CtIP − Ctinput) and the ratio of IP RNA template and input RNA template to initial RNA when reverse transcription is introduced. Detailed primers are shown in supplementary Table 1 (Table S1 ).
Evo m mlv rt kit with gdna clean for qpcr kit
Manufactured by Accurate Biology
Sourced in China
The Evo M-MLV RT Kit with gDNA Clean for qPCR is a laboratory product designed for reverse transcription and genomic DNA (gDNA) clean-up prior to quantitative PCR (qPCR) analysis. The kit provides the necessary reagents and components to perform these specific functions.
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3 protocols using evo m mlv rt kit with gdna clean for qpcr kit
1
Investigating m6A Modifications in Queshan Black Pigs
2
RNA Extraction and qRT-PCR Analysis
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RNA was extracted by the TRIzol reagent (Invitrogen, Carlsbad, CA) and 500μg of RNA was reverse transcribed to cDNA using Evo M-MLV RT Kit with gDNA Clean for qPCR kit (Accurate Biology, Changsha, China). Real-time RT-PCR was performed with SYBR Green Premix Pro Taq HS qPCR Kit (Accurate Biology, Changsha, China) using the QuantStudio 5 Real Time PCR system (Applied Biosystems). The qRT-PCR primers for TOP2A are listed in S2 Table in S1 File .
3
Quantitative Real-Time PCR Protocol
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Total RNA was extracted and purified from cultured cells using the standard TRIZOL RNA extraction protocol (AG, AG21102). First-strand cDNA was synthesized from 1 µg of RNA using the Evo M-MLV RT Kit with gDNA Clean for qPCR Kit (Accurate Biology, AG11705). Briefly, 20-µl reactions were prepared by combining 10 µl of reaction mix, 1 µl of Evo M-MLV RTase Enzyme Mix, 1 µl of RT Primer Mix, 4 µl of 5 × RTase Reaction Buffer Mix I, and 4 µl of RNA-free water. The cDNA from various cell samples was then amplified by realtime qPCR with specific primers using the SYBR Green Premix Pro Taq HS qPCR Kit (Accurate Biology, AG11701). The gene expression was calculated via the 2−ΔΔCt method and normalized to RNA18S. The relative concentrations of mRNA were expressed in arbitrary units based on the untreated group, which was assigned a value of 1. The primers, which were synthesized and desalted from Sigma-Aldrich, are shown in Table 1 .
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