Mx3005p real time pcr system

Total RNAs were extracted using RNAprep pure plant kit (TIANGEN, Beijing, China) from different rubber tree materials, and 1 μg of total RNA was used for first-strand cDNA synthesis. The gene-specific primers for qRT-PCR were designed using the software Primer Primer 5.0 (Davis, CA, USA) according to HbCPK gene sequences listed in Supplementary Table S1. Rubber tree actin gene (GenBank Acc. HQ260674.1) was used as an internal control. Quantitative real-time PCR (qRT-PCR) was conducted using Mx3005P real-time PCR system with SYBR green master mix (2X) (Thermo Fisher Scientific, Foster, CA, USA) in accordance with previously described PCR conditions [36 (link)]. Data were processed using 2^−ΔΔCt methods, and the relative expression level of each CPK gene were used to generate a heat map using MultiExperiment viewer software (MeV, version 4.9, Boston, MA, USA). Semi-quantitative PCR assays were performed using the same cDNA templates and primers used for qRT-PCR. The expression levels of each HbCPK gene were visualized using agarose gel electrophoresis of the corresponding PCR products.

Cells were dissolved in the TRIzol reagent (Invitrogen, 12044977) for total RNA extraction. RNA quality was confirmed by optical density measurement. cDNA was synthesized from 1 μg of total RNA using random primers and Moloney murine leukemia virus reverse transcriptase (MultiScribe, Applied Biosystems, 10117254). The cDNA was used for qRTPCR in an Mx3005P Real-Time PCR System (ThermoFisher Scientific). Reactions were carried out in duplicate for all conditions using a Sybr Green Master mix (ThermoFisher Scientific, 4344463) or Fast Taqman mix (ThermoFisher Scientific, 4444557) and expression of mouse Gapdh mRNA (Life Technology, 433764T) was used as endogenous control in the comparative cycle threshold method (2⁻ΔΔ^Ct). Primer sequences used are listed in Supplementary Table S2.

Total RNA was extracted from frozen tumors, lungs, and cultured cells with TRIzol (Invitrogen, 12044977). cDNAs (synthesized using random primers and Moloney murine leukemia virus reverse transcriptase (MultiScribe, Applied Biosystems, 10117254)) were used for qRT–PCR in an Mx3005P Real‐Time PCR System (Thermo Fisher Scientific) with Sybr Green Master mix (Thermo Fisher Scientific, 4344463) or Fast Taqman mix (Thermo Fisher Scientific, 4444557). Expression of mouse Gapdh mRNA (Life Technology, 433764T) was used as endogenous control in the comparative cycle threshold method (2‐ΔΔC_t) with the listed primers (Appendix Table S7).