Total RNA was extracted from cultured cells with TRIZOL reagent (Invitrogen), according to the manufacturer’s protocol. All cDNA samples were prepared using an All-in-one First-stand cDNA synthesis kit (GeneCopoeia, MD, USA). Quantitative real-time PCR (RT-qPCR) analyses were performed with an All-in-one qPCR mix (GeneCopoeia), according to manufacturer’s instructions using an ABI StepOnePlus™ qPCR system. Primers and gene ID are listed in Additional file 1 : Table S2.
All in one first stand cdna synthesis kit
Manufactured by GeneCopoeia
Sourced in United States
The All-in-one First-strand cDNA Synthesis Kit is a ready-to-use reagent system designed for the reverse transcription of RNA to complementary DNA (cDNA). The kit contains all the necessary components, including reverse transcriptase, buffer, and dNTPs, to efficiently convert RNA into cDNA in a single reaction.
Automatically generated - may contain errors
3 protocols using all in one first stand cdna synthesis kit
1
RNA Extraction and qPCR Analysis Protocol
2
Quantitative RT-qPCR Analysis of VEGF and REC8
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Total RNA was extracted from cultured cells with trizol reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s protocol. All cDNA samples were prepared using an All-in-one First-stand cDNA synthesis kit (GeneCopoeia, MD, USA). Quantitative real-time PCR (RT-qPCR) analyses were performed with an all-in-one qPCR mix (GeneCopoeia) according to manufacturer’s instructions using an ABI StepOne-Plus™ qPCR system. The primer for VEGF: Forward: 5′-TGCAGATTATGCGGATCAAACC-3′; Reverse: 5′-TGCATTCACATTTGTTGTGCTGTAG-3′; REC8: Forward: 5′-CATCCCACCAGAAGAACGG-3′; Reverse: 5′-GCACCAAAGGCATCTCCAT-3′; β-actin: Forward 5′-ATCGTGCGTGACATTAAGGAGAAG-3′; reverse:5′-AGGAAGGAAGGCTGGAAGAGTG-3’.
3
Quantifying Gene Expression in NPC Tissues
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The total RNA in NPC tissue, chronic nasopharyngeal mucosal in ammatory tissue and S18, 6-10B cells were extracted by Trizol (Takara Co., Ltd., Dalian, China). The instructions of the All-in-One First-Stand cDNA Synthesis Kit (GeneCopoeia, Maryland, USA) was applied to perform reverse transcription of RNA into cDNA. Designed primers including HOTAIR, EZH2 and E-cadherin were synthesized by Shanghai Genechem Co., Ltd. (Shanghai, China) (Table 1). The mRNA expression of each gene was detected by a real-time PCR kit (Takara), and detected using a real-time PCR instrument (ABI 7500, ABI, Foster City, CA, USA) with glyceraldehyde phosphate dehydrogenase (GAPDH) as an internal reference. The 2 -ΔΔCt referred to the calculation of the relative expression of each target gene. Each experiment was repeated 3 times.
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