Total RNA extraction was performed using the TRIzol reagent (Life Technologies, Rockville, MD, USA), according to the manufacturer's instructions. Amplification of transcripts was performed by reverse transcriptase polymerase chain reaction kit (Life Technologies). PCR amplification was performed using the following primers: Mcl-1, forward: 5′- GCG ACT GGC AAA GCT TGG CCT CAA-3′, reverse: 5′- GTT ACA GCT TGG ATC CCA ACT GCA -3′.
Reverse transcriptase polymerase chain reaction kit
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Reverse Transcriptase Polymerase Chain Reaction (RT-PCR) kit is a laboratory tool used for the detection and quantification of RNA molecules. It combines the reverse transcription of RNA into complementary DNA (cDNA) with the subsequent amplification of the cDNA using the polymerase chain reaction (PCR) technique. This kit provides the necessary reagents and enzymes to perform this two-step process, enabling the analysis of RNA expression levels in various biological samples.
Automatically generated - may contain errors
5 protocols using reverse transcriptase polymerase chain reaction kit
1
Extracting and Amplifying Total RNA
2
Quantitative RT-PCR for Mcl-1 Expression
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Total RNA was extracted by using TRIzol reagent (Life Technologies). Amplification of transcripts was performed by reverse transcriptase polymerase chain reaction kit (Life Technologies). RT-PCR was performed on an Applied Biosystems 9700 RT-PCR using gene-specific oligonucleotide primer for Taqman probes (Applied Biosystems). Taqman probes were as follows: GAPDH (Hs99999905_m1), Mcl-1 (HS01050896_m1). For expression of mRNA, gene expression was normalized by the GAPDH.
3
Gene Expression Analysis in Hypoxia
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RNA was extracted using the TRIzol reagent (Life Technologies, Grand Island, NY, USA). Transcripts were amplified using a reverse transcriptase polymerase chain reaction kit (Life Technologies). Real-time PCR was performed on an Applied Biosystems 9700 real-time PCR system using gene-specific oligonucleotide primers for TaqMan probes (Applied Biosystems, Foster City, CA, USA). TaqMan probes were as follows: GAPDH (Hs99999905_m1), HIF-1α (Hs00153153_m1), and vascular endothelial growth factor (VEGF, Hs00900055_m1). For mRNA expression, gene expression was normalized t GAPDH.
4
Quantitative mRNA Expression Analysis
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Total RNA was extracted using the TRIzol reagent (Life Technologies). Amplification of the transcripts was performed by reverse transcriptase polymerase chain reaction kit (Life Technologies, Carlsbad, CA, USA). The PCR was performed on Applied Biosystems 9700 RT-PCR using gene-specific oligonucleotide primers and Taqman probes (Applied Biosystems, Foster City, CA, USA). The probes were as follows: Glyceraldehyde-3-Phophate Dehydrogenase (GAPDH) (Hs99999905_m1), XIAP (Hs00745222_s1). For mRNA quantification, gene expression was normalized by GAPDH. Relative expression levels were calculated using the 2(-Delta Delta C(T) method [20 (link)].
5
Quantitative Gene Expression Analysis
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Total RNA was extracted by using TRIzol reagent (Life Technologies). Amplification of transcripts was performed using a reverse transcriptase polymerase chain reaction kit (Life Technologies). Real-time PCR was performed using the QuantStudio 6 Flex system. Taqman probes were as follows: STAT3 (Hs00374280_m1), GLI-1 (Hs01110766_m1), EPHB3 (Hs00177903_m1) and GAPDH (Hs99999905_m1). GAPDH was used for the normalization of gene expression.
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