Rt amv avian myeloblastosis virus transcriptase

For quantitative real time PCR (qRT-PCR) analysis, total RNA from leaf tissues or stem tissues was extracted using RNA RNeasy Plant Mini Kit (Qiagen, Hilden, Germany) and treated with RNase-free DNase (TaKaRA, Dalian, China). Samples from at least three plants were pooled for analysis. The quality or integrity of RNA was checked by agarose gel electrophoresis and P100 spectrophotometer (Pultton, Ann Arbor, MI, USA). The criteria of high-quality total RNA include the following: (1) sharp distinct 28S and 18S rRNA bands, with the 28S band approximately twice as intense as the 18S band; (2) the value of D260/OD280 between 1.9 and 2.0; and (3) no detected genomic DNA band. The qualified RNA samples were reversely transcribed using RT-AMV (Avian Myeloblastosis Virus) transcriptase (TaKaRa, Dalian, China). Subsequently, qRT-PCR was performed in a volume of 20 μL containing 10 μL of SYBR Premix ExTaq TM (TaKaRa, Dalian, China). ACTIN7 and UBQ were used as an internal control in Arabidopsis and Populus, respectively. Three biological replicates of each sample and three technical replicates of each biological experiment were conducted. Primers used for qRT-PCR were listed in Table S1.

For quantitative real time PCR (qRT-PCR) analysis, total RNA from fresh tissues was extracted using RNA RNeasy Plant Mini Kit (Qiagen, Hilden, Germany) and treated with RNase-free DNase (TaKaRA, Dalian, China). Samples from at least three plants were pooled for analysis. The quality or integrity of RNA was checked by agarose gel electrophoresis and P100 spectrophotometer (Pultton, Ann Arbor, MI, USA). The criteria of high-quality total RNA include the following: (1) sharp distinct 28S and 18S rRNA bands, with the 28S band approximately twice as intense as the 18S band; (2) the value of D260/OD280 between 1.9 and 2.0; and (3) no detected genomic DNA band. The qualified RNA samples were reversely transcribed using RT-AMV (Avian Myeloblastosis Virus) transcriptase (TaKaRa, Dalian, China). PCR amplification was performed for CDS of a random gene containing an intron to exclude DNA contamination. Subsequently, qRT-PCR was performed in a volume of 25 μL containing 12.5 μL of SYBR Premix ExTaq TM (TaKaRa, Dalian, China). ACTIN2 rRNA was used as an internal control. Five biological replicates of each sample and three technical replicates of each biological experiment were conducted. Primers used for qRT-PCR were listed in Table S1.