Total RNA from cells were extracted using Trizol reagent (Life Technologies, Benicia, CA, USA). RT–PCR was performed using SuperScript One-Step RT–PCR Platinum Taq system (Life Technologies) with 50 ng RNA and one of the following primer pairs CK1/CK1R, CK2/CK2R, CK3/CK3R or CK1/CK3R (See Supplementary Information ). RT–PCR products were separated on agarose gel, purified and re-amplified by PCR. PCR products were cleaned with ExoSAP-IT PCR product cleanup reagent (Affymetrix) and sequenced in both strains using BigDye Terminator 3.1 cycle sequencing kit (Life Technologies) as described (Ren et al, 2013 (link)). Quantitative RT–PCR (RT–qPCR) was performed using CK2/CK2R or CK-T/CK-R primer pairs (Supplementary Information ). RT–qPCR was performed using Power SYBR Green reagents (Applied Biosystems, Foster City, CA, USA). Samples were assayed in triplicates as described (Schneeberger et al, 2014 (link)).
Superscript one step rt pcr platinum taq system
Manufactured by Thermo Fisher Scientific
The SuperScript One-Step RT–PCR Platinum Taq system is a laboratory equipment product designed for reverse transcription and polymerase chain reaction (RT-PCR) in a single-step process. It includes reagents for both the reverse transcription and amplification steps, enabling efficient and streamlined RNA analysis.
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2 protocols using superscript one step rt pcr platinum taq system
1
RT-PCR and qRT-PCR Analysis of RNA
2
Reverse Transcription-PCR for SHP2 Gene
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RNA was extracted from frozen tissues using Trizol reagent (Life Technologies). Samples were treated with DNase I to avoid DNA contamination and RT-PCR was performed using the SuperScript One-Step RT-PCR Platinum Taq system (Life Technologies) with the following primers: SHP2F1: 5′-GGTTGGACAAGGGAATACGG-3′ and SHP2R2: 5′-AGGGCTCTGATCTCCACTCG-3′. The protocol for a 50 μl RT-PCR reaction was: 30 min cDNA synthesis at 55 oC, 4 min denaturation at 94 oC then 35 cycles of 94 oC for 30 sec, 57 oC for 30 sec, then 72 oC for 30 sec with a final extension step of 72oC for 4 min which yields a 462-bp fragment.
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