Mmlv reverse transcription pcr kit with oligo dt 15 rt pcr

Total RNA extraction was performed using the cetyltrimethylammonium bromide-based extraction as described [49 (link)]. Complementary DNAs were synthesized using the MMLV Reverse transcription PCR Kit with oligo(dT)15 (RT-PCR, Evrogen, Moscow, Russia) as described [50 (link)]. qRT-PCRs were performed using the real-time PCR kit (Evrogen) and SybrGreen I Real-time PCR dye (Evrogen) using total cDNAs as described [49 (link),50 (link)].
The expression was calculated by the 2^−ΔΔCT method with two internal controls VaGAPDH and VaActin1, as described [51 (link)]. The qRT-PCR primers are listed in the Table S2. We used different primer sets for analyzing expression of the exogenous (transgene) and endogenous VaMyb genes (Supplementary Figure S3; Table S2).

Total RNA extraction was performed using the cetyltrimethylammonium bromide-based extraction as described [47 (link)]. Complementary DNAs were synthesized using the MMLV Reverse transcription PCR Kit with oligo(dT)15 (RT-PCR, Evrogen, Moscow, Russia) as described [60 (link)]. qRT-PCRs were performed using the real-time PCR kit (Evrogen) and EvaGreen Real-time PCR dye (Biotium, Hayward, CA, USA) using total cDNAs as described [28 (link),46 (link)]. The expression was calculated by the 2^−ΔΔCT method with two internal controls, VaGAPDH and VaActin1, as described [61 (link)]. The primers used for qRT-PCRs are listed in Table S1. We used different primer sets for analyzing expression of the exogenous (transgene) and endogenous VaCML genes. To analyze transcript levels of the VaCML transgenes, the primers were designed according to the promoter and terminator sequences of the transgenes (Figure S4; Table S1). To analyze expression of the endogenous VaCML genes, the primers were designed to the untranslated (UTR) and specific coding gene regions of the VaCMLs (Figure S4; Table S1). To analyze transcript levels of both VaCML transgenes and VaCML endogenes (total expression), the primers were designed to protein-coding regions of VaCML genes (Figure S4; Table S1).

RNA was extracted by the CTAB-based method as described [64 (link)]. cDNAs were produced using the MMLV Reverse transcription PCR Kit with oligo(dT)15 (RT-PCR, Evrogen, Moscow, Russia) as described [65 (link)].
The mRNA transcript levels of the transgenes were determined by real-time quantitative PCR (qRT-PCR)—the 2^−ΔΔCT method [66 (link)] with two internal controls, including AtGAPDH (NM_111283.4) and AtEF (XM_002864638) [65 (link)]. The primers designed for qRT-PCRs are shown in Tables S1 and S2.
qRT-PCR reactions were performed in volumes of 20 µL using the real-time PCR kit (Evrogen), as described [65 (link)], containing 1x Taq buffer, 2.5 mM MgCl₂, 0.2 mM of each dNTP, 0.2 µM of each oligonucleotide primer, 1x SybrGreen I Real-time PCR dye, 1 µL cDNAs, and 1 unit of Taq DNA polymerase (Evrogen). Analysis was performed in DTprime 4M1 Thermal Cycler (DNA-technology, Moscow, Russia) programmed for an initial denaturation step of 2 min at 95 °C followed by 50 cycles of 10 s at 95 °C and 25 s at 62 °C.