Total RNA was isolated from cells using TRI Reagent (Invitrogen) according to the manufactures protocol. To synthesize cDNA, 2 μg of RNA was subjected to DNase treatment and incubated for 15 min at room temperature. The RNA was then reverse transcribed into cDNA using Superscript III first strand RT-PCR system and random hexamers (Invitrogen). The cDNA product was subjected to PCR using the forward primer 5′-CAGAGGGCACAGGAAAGAAG-3′ and the reverse primer 5′-GAACTAGGATTCGGGCAACA-3′ for the detection of human SESTRIN 2. GAPDH was used as an endogenous control using the forward primer 5′-ACCACAGTCCATGCCATC-3′ and reverse 5′-TCCACCACCTGTTGCTG-3′.
Superscript 3 first strand rt pcr system
Manufactured by Thermo Fisher Scientific
The Superscript III first strand RT-PCR system is a reverse transcription-polymerase chain reaction (RT-PCR) kit designed for the synthesis of first-strand cDNA from total RNA or poly(A)+ RNA. It includes the SuperScript III Reverse Transcriptase enzyme, which provides high sensitivity and efficiency in cDNA synthesis.
Automatically generated - may contain errors
2 protocols using superscript 3 first strand rt pcr system
1
Quantification of SESTRIN 2 Expression
2
Murine Atg7 Expression Analysis
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Total RNA was isolated using Trizol (Invitrogen, 15596018) according to the manufacturer's instructions. For cDNA synthesis, 2 μg of RNA was subjected to DNase treatment followed by EDTA inactivation and the RNA reverse transcribed into cDNA using Superscript III first strand RT-PCR system and random hexamers (Invitrogen, 18080–051) according to the manufacturer's instructions. The cDNA product was subjected to 25 cycles of PCR using the forward primer 5′-TTCCAAGGTCAAAGGACAAA-3′ and the reverse primer 5′- ACAGCTTTAGGACAATCT-3′ for the detection of murine Atg7. Gapdh was used as an endogenous control using the forward primer 5′-ACCACAGTCCATGCCATC-3′ and reverse 5′-TCCACCACCTGTTGCTG-3′.
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