Tb green premix ex taqtm 2 tli rnaseh plus kit

Total RNA was extracted from the whole leaves of plants that were either B. cinerea-infected or mock-treated, by using the RNAiso Plus kit (Takara, Beijing, China) and following the manufacturer’s instructions. Quality and integrity of RNA were assessed by gel electrophoresis and A₂₆₀/A₂₈₀ and A₂₆₀/A₂₃₀ ratios in a NanoDrop photometer (PeqLab, Germany). Pure and highly intact RNA samples were used for cDNA synthesis with Hifair^TM 1st Stranded cDNA Synthesis SuperMix for qPCR (gDNA digester plus) kit (Yeasen, Shanghai, China). The generated cDNA was then subjected to quantitative PCR (qPCR) with gene-specific primers (Supplementary Table S3), by using the TB Green^® Premix Ex Taq^TM II (Tli RNaseH Plus) kit (Takara). The qPCRs were implemented in a CFX Connect Real-Time System (Bio-Rad, United States), with three technical replicates in the same run with at least three biological replicates used. For normalization purposes, EXP (At4g26410) was used as an endogenous reference gene because it has high expression stability under varying plant stress conditions (Czechowski et al., 2005 (link); Liu S. et al., 2017 (link)). Primers used for reference genes and genes of interest are listed in Supplementary Table S3. The ΔΔCt method (Livak and Schmittgen, 2001 (link)) was used to calculate the relative expression levels of genes.

The mRNA levels of FL-SMN, SMNΔ7 and GAPDH were quantified using TB Green^® Premix Ex Taq^TM II (Tli RNaseH Plus) Kit (TaKaRa, Kyoto, Japan) and the following primers: FL-SMN2 forward primer (5′-GCTCACATTCCTTAAATTAAGGAGAAA-3′), SMN2 Δ7 forward primer (5′-TGGCTATCATACTGGCTATTATATGGAA-3′) and SMN2 reverse primer (5′-TCCAGATCTGTCTGATCGTTTCTT-3′) for the amplification of endogenous FL-SMN and SMNΔ7 mRNAs, GAPDH forward primer (5′-CAACGGATTTGGTCGTATTGG-3′) and GAPDH reverse primer (5′-TGATGGCAACAATATCCACTTTACC-3′) for GAPDH mRNAs. Real-time PCR was carried out at the following temperatures for indicated times: Step 1: 48 °C (15 min); Step 2: 95 °C (10 min); Step 3: 95 °C (15 s); Step 4: 60 °C (1 min); Steps 3 and 4 were repeated for 40 cycles. The Ct values for each mRNA were converted to mRNA abundance using actual PCR efficiencies. The mRNA levels of FL-SMN and SMNΔ7 were primarily normalized to that of GAPDH, and relative amount of each mRNAs was presented as a percentage of DMSO-treated SMA fibroblasts.

Total RNA was extracted using RNAprep Pure Plant Plus Kit (for polysaccharides and polyphenolic-rich samples) (TIANGEN, Beijing, China) according to the manufacturer’s instructions. RNA purity and concentration were determined using a NanoDrop spectrophotometer (Thermo Scientific, Waltham, MA, USA). RNA integrity was evaluated using agarose gel electrophoresis. Reverse transcription was performed using PrimeScript^TM RT Reagent Kit with gDNA Eraser (Takara, Kusatsu, Japan) and real-time fluorescence quantitative PCR (RT-qPCR) was conducted using TB Green Premix Ex Taq^TM II (Tli RNaseH Plus) kit (Takara, Kusatsu, Japan) on a 7500 Real-Time PCR System (Applied Biosystems, Waltham, MA, USA). PpACTIN7 was used as the internal reference. Three biological repeats were performed for each sample and the relative expression levels of genes were calculated by the 2^−ΔΔCt method [46 (link)]. Primers used for RT-qPCR are shown in Supplemental Table S1.

The total RNA (1 μg) extracted from the cells on day 6 of the maturation phase using acid guanidium thiocyanate/phenol/chloroform was reverse transcribed (RT) using M-MLV reverse transcriptase (Ribonuclease H Minus Point Mutant). The single-stranded cDNA was synthesized using oligo-(dT)₁₅ and a random 9-mer (Promega Corp., Madison, WI, USA) as primers in the RT reaction. The amount of transcript was determined via qRT-PCR using TB GreenTM Premix Ex TaqTM II (Tli RNaseH Plus) kits (Takara Bio Co., Inc., Kusatsu, Japan) and a Thermal Cycler Dice^TM Real Time System (Takara Bio Co., Inc., Kusatsu, Japan) according to the threshold cycle (CT) and ^ΔΔCT methods described by the manufacturer. Table 1 shows the oligonucleotides used herein. The cycling program comprised 95 °C for 30 s, 40 cycles at 95 °C for 5 s and 60 °C for 30 s, followed by 95 °C for 15 s and 60 °C for 30 s. Amounts of target gene transcripts were normalized to those of β-Actin. The accession numbers of the target genes are as follows: PPARγ, NM_011146; adiponectin, NM_009605.5; LPL, NM_008509.2; DP1, NM_008962.4; DP2, XM_006526696.5; β-Actin, NM_007393.

Total RNA (1 μg) extracted from the cells after 6, 24, and 48 h of the differentiation phase, and on day 6 of the maturation phase using acid guanidium thiocyanate/phenol/chloroform was reverse transcribed (RT) using M-MLV reverse transcriptase (Point mutation without Ribonuclease H activity). Single-stranded cDNA was synthesized using oligo-(dT)₁₅ and a random 9-mer (Promega) as primers in the RT reaction. Transcript levels were determined by RT-qPCR using TB GreenTM Premix Ex TaqTM II (Tli RNaseH Plus) kits (Takara Bio Co., Inc., Kusatsu, Japan) and a Thermal Cycler Dice^TM Real Time System (Takara Bio Co., Inc.) according to the threshold cycle (CT) and ^ΔΔCT methods described by the manufacturer. Table 1 shows the oligonucleotides used herein. The cycling program comprised 95 °C for 30 s, 40 cycles at 95 °C for 5 s and 60 °C for 30 s, followed by 95 °C for 15 s and 60 °C for 30 s. Levels of target gene transcripts were determined and normalized to those of β-Actin. The accession numbers of the target genes are as follows: C/Ebpβ, NM_009883; C/Ebpδ, NM_007679; Pparγ, NM_011146; C/Ebpα, NM_001287523; Lpl, NM_008509; Glut4, AB008453; Leptin, NM_008493; β-Actin, NM_007393.