cDNA was reverse transcribed (30?μl), and experiments were performed following the procedure recommended by TaKaRa M-MLV reverse transcriptase (Takara Bio, Inc.) product specifications. Quantitative reverse transcription-polymerase chain reaction (qRT-PCR) experiments were performed according to the recommended methods provided by the manufacturer of the TaKaRa SYBR® Premix Ex Taq kit (Takara Bio, Inc.). U6 and GAPDH were used as housekeeping genes for standardization. The primer sequences are listed in Supplementary Table 1 . Data are presented as the fold change of downregulation or upregulation (fold value = 2-ΔΔCt, where ΔΔCt = (Ct of the gene of interest, treated-Ct of the housekeeping gene, treated) - (Ct of the gene of interest, control-Ct of the housekeeping gene, control), and Ct was the number of threshold cycles).
Takara m mlv reverse transcriptase
Manufactured by Takara Bio
TaKaRa M-MLV reverse transcriptase is an enzyme used for the conversion of RNA to complementary DNA (cDNA). It catalyzes the synthesis of single-stranded cDNA from an RNA template.
Automatically generated - may contain errors
Lab products found in correlation
Trizol, by Thermo Fisher Scientific (2 mentions)
Steponeplus real time pcr system, by Thermo Fisher Scientific (2 mentions)
Takara sybr premix ex taq kit, by Takara Bio (1 mentions)
Rna free water, by Applygen (1 mentions)
Sybr premix ex taqtm kit, by Takara Bio (1 mentions)
La taq polymerase, by Takara Bio (1 mentions)
Peasy t vector, by Transgene (1 mentions)
Rnaiso plus kit, by Takara Bio (1 mentions)
Kapa sybr rapid quantitative pcr kit, by Roche (1 mentions)
4 protocols using takara m mlv reverse transcriptase
1
Reverse Transcription and qRT-PCR Analysis
2
Quantitative gene expression analysis
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RNA from cell samples was extracted using Trizol (Thermo Fisher Scientific) following the manufacturer’s instructions. RNA was eluted in 30 μL RNA-free water (Applygen), aliquoted, reverse transcribed (60 μL), and treated according to the protocol recommended for the TaKaRa M-MLV reverse transcriptase (Takara). Amplification of the gene fragment was performed as follows: 72 °C for 60 min, 42 °C for 10 min. Real-time PCR experiments were performed following the manufacturer’s procedure for the Takara SYBR® Premix Ex TaqTM kit. To amplify the UCP1, PPARγ, PGC-1a, TRPC3, PPARa, and GAPDH genes, one-step RT-PCR was performed. Amplification of gene fragments was performed as follows: 95 °C for 5 min, 95 °C for 10 s, 60 °C for 40 s, 40 cycles. Reactions were performed in triplicate, and independent experiments were repeated three times. The RT-PCR data were analyzed using StepOne Software 2.1.
3
Identification of Putative LACS Genes in Chlamydomonas
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The putative LACSs genes in C. reinhardtii were screened from NCBI genome database using nucleotide sequence from A. thaliana, N. oculata and T. pseudonana by BLAST. Primers listed in Additional file 1 : Table S1 were designed according to BLAST results. Total RNA was isolated by Takara RNAiso Plus Kit according to its instruction. With Takara Reverse Transcriptase M-MLV, the first string of cDNA was synthesized using oligo-dT as the reversed primer according to its protocol. Cracs1 and cracs2 were amplified using LA Taq polymerase (Takara) with high GC buffer II by primer pairs crACSF1/crACSR1 and crACSF2/crACSR2, respectively. PCR products were then cloned into pEASY-T vector (Transgene) after purification and subjected to sequencing. Open reading frame (OFR) was predicted using ORF Finder (http://www.ncbi.nlm.nih.gov/gorf/gorf.html ). Protein sequences were aligned using ClustalX and phylogenetic tree was constructed using MEGA 4.0.
4
Analysis of Rose Petal Transcriptome in Botrytis Infection
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RNA-Seq data from rose petals infected with B. cinerea were obtained from the National Center for Biotechnology Information (NCBI) database (accession number PRJNA414570). Clean sequencing reads were mapped to the rose reference genome, and the number of reads per kb per million reads (RPKM) were used to determine the gene expression levels. To confirm the RNA-Seq results, the transcript abundance of 6 RcWRKY genes was analyzed using qRT-PCR. To this end, cDNA was generated from rose petals inoculated with B. cinerea, using Takara Reverse Transcriptase M-MLV (Takara). Quantitative RT-PCR was performed on a StepOnePlus Real-Time PCR System (Thermo Fisher Scientific), by using 1 μL of the first strand cDNA in the reaction with the KAPA SYBR rapid quantitative PCR kit (KAPA Biosystems). RhUbi was used as a housekeeping gene. The primers used for determining transcript abundance are listed in Additional file 3 : Table S2.
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