Sybr premix ex taq tli rnaseh plus

Total RNA extraction from both oviposited and non-oviposited pods of mild tolerant black gram variety were performed with PureLink^™ Plant RNA reagent (Ambion) and treated with DNAse-I (Sigma-Aldrich, USA) to eliminate traces of genomic DNA. The cDNA was synthesized using a PrimeScript^™ RT Reagent Kit with gDNA Eraser (Perfect Real Time) (Clontech,USA) and real time PCR protocol was followed according to manufacturer’s instructions given in SYBR^® Premix ExTaq^™ (Tli RNAse H Plus) (Clontech,USA). The thermal cycling conditions were carried out in Applied Biosystems StepOnePlus^™ Real-Time PCR System (Applied Biosystems, USA) using the following program- 95°C for 30 sec followed by 40 cycles of 95°C for 5 sec and 60°C for 30 sec followed by a melt curve stage at 60°C for 1 min. Gene-specific primers were designed using Oligo Perfect Designer software program having a GC content of 55–60%, a T_m>50°C, primer length ranging from 18–22 nucleotides and an expected amplicon size of 100-150bp. All quantitative real-time PCR experiments were performed twice using two biological replicates and each reaction was run in triplicate using the designed gene specific primers (S1 Table).
The relative gene expression levels were obtained by relative quantification (RQ) according to the 2^-ΔΔCt method [16 (link)].

Total RNA was extracted from cells using TRIzol (Thermo Fisher Scientific) according to the manufacturer’s instructions. cDNA was synthesized from the total RNA using the Maxima Reverse Transcriptase enzyme, random hexamer primers, dNTP mix, and the Ribolock RNase inhibitor (Thermo Fisher Scientific). PCR was performed on the template cDNA using Phusion High-Fidelity DNA Polymerase and dNTP mix (Thermo Fisher Scientific). Quantitative PCR was alternatively performed for the cDNA using the SYBR Premix Ex Taq (Tli RNase H Plus) (TAKARA-Clontech) using a QuantStudio5 system (Applied Biosystems). The primer sequences used for these experiments were synthesized by Eurogentec and are shown in Table S4.

Epidermis samples were collected from every three silkworms after 6 hours of molting in the 4^th instar, and total RNA was prepared using RNAiso Plus and reverse transcribed using the Reverse Transcriptase M-MLV (RNase H-) kit (TaKaRa, China) after treatment with DNase. The cDNA was diluted to 20 ng/μL and used as the template for qRT-PCR. The 20-μL reaction included 1 μL primer (10 μmol/L, S1 Table), 1 μL cDNA, 10 μL 2×SYBR^® Premix Ex Taq^™ (Tli RNaseH Plus) (TaKaRa, China) and 8 μL ddH₂O. After a rapid centrifugation step, quantitative reverse transcription PCR (qRT-PCR) was performed using a LightCycle 96 real time PCR system (Roche, Switzerland) with the reaction program below: a three-step reaction protocol of 45 cycles of 95°C for 10 s, 58°C for 10 s and 72°C for 10 s after a 10-min step of pre-degeneration, followed by melting. Relative expression was calculated using the 2^–ΔΔCt method [25 (link)] with glyceraldehyde-3-phosphate dehydrogenase (GAPDH, BGIBMGA007490) as the reference gene. Melting analysis was performed to guarantee the qRT-PCR quality. The relative expression of 932VR and q-l^p were compared, and RNA-Seq and qRT-PCR data were compared to analyze several key pathways.

Partial archaeal amoA genes detected in the throughfall and leaf samples were quantified by qPCR (LightCycler Nano; Roche, Basel, Schweitz) using archaeal amoA gene-specific primers (amoA19F and amo643R). The reaction was conducted in a 20-µL reaction mixture containing 3.8 µL of distilled H₂O, 5 µL containing 0.1–2 ng of template DNA, 0.4 µL of 10 µM each primer, 0.4 µL of bovine serum albumin (20 mg mL⁻¹; Takara Bio Inc.), and 10 µL of 2× SYBR Premix Ex Taq (Tli RNaseH Plus) (Takara Bio Inc.). The template DNA concentration was measured using a Qubit 2.0 fluorometer and a Quant-iT dsDNA HS assay kit (Thermo Fisher Scientific) in accordance with the manufacturer’s instructions. The thermal cycling conditions were as follows for the archaeal amoA gene: 95 °C for 30 s; 45 cycles consisting of 95 °C for 30 s, 55 °C for 1 min and 72 °C for 40 s; final extension at 72 °C for 10 min with a melting curve analysis.
The archaeal amoA gene was amplified from an environmental clone, N3-16 (AB622272), using the amoA19F and amo643R primers. The PCR products were purified using a QIAquick PCR purification kit (Qiagen). This amplicon was used for construction of a standard curve of qPCR. qPCR analysis of each sample was conducted in triplicate, and the efficiency of the qPCR reaction was 86.6–87.5 %. The quantitative lower limit for the archaeal amoA gene was 44 copies per reaction.

The heads of nm2 mutant and wildtype C603 larvae in the pre-molting stage were collected. Total RNA was extracted using RNAiso Plus (TaKaRa, China), according to the instructions of the manufacturer. After treatment with DNase I (TaKaRa, China), cDNA was synthesized using Reverse Transcriptase M-MLV (RNase H-) kit (TaKaRa, China), according to the instructions of the manufacturer, and was then diluted to 50 ng/μL to serve as the template for qRT-PCR. The 20-μL reaction system included 1 μL of primers (10 μmol/L, S1 Table), 1 μL of cDNA, 10 μL of 2×SYBR^® Premix Ex Taq™ (Tli RNaseH Plus) (TaKaRa, China) and 8 μL of ddH₂O. After transient centrifugation, qRT-PCR was performed using a LightCycle 96 Real-time PCR System (Roche, Switzerland) with the following reaction program: three-step reaction protocol consisting of 45 cycles of 95°C for 10 s, 58°C for 10 s and 72°C for 10 s, after a 10-min step of pre-degeneration, followed by melting. The quality of the qRT-PCR product was tested through melting curve analysis, and relative expression was calculated using the 2^–ΔΔCt method [19 (link)], with the average of three house-keeping genes, RPL3 (BGIBMGA013567), GAPDH (BGIBMGA007490) and BmActin3 (BGIBMGA005576), serving as the reference. The qRT-PCR results were compared with the DGE results to verify the accuracy of the DGE data.

Expression profiling of AlOBPs and AlCSPs was performed using quantitative real time-PCR (qRT-PCR) performed in a LightCycler^® 96 (Roche, Switzerland) with a mixture of 5 µL 2X SYBR^® Premix Ex Taq (Tli RNaseH Plus) (TaKaRa, Dalian), 0.2 µL of each primer (10 µM), 2.5 ng of sample cDNA, and 3.6 µL of sterilized ultrapure H₂O. The reaction program was as follows: 30 s at 95 °C, 40 cycles of 95 °C for 5 s, and 60 °C for 20 s. The results were analyzed using a LightCycler^® 96 SW 1.1. The qRT-PCR primers (Table S2) were designed with Beacon Designer 7.9 (PREMIER Biosoft International, CA, USA). This was followed by the measurement of fluorescence over a 55 to 95 °C melting curve to detect a single gene-specific peak and to check the absence of primer dimer peaks, and a single and discrete peak was detected for all primers tested. Negative controls consisted of non-template reactions where the cDNA was replaced with H₂O.
Expression levels of AlOBPs and AlCSPs were calculated relative to the reference genes AlGAPDH (A. lepigone glyceraldehyde-3-phosphate dehydrogenase) and AlEF (A. lepigone elongation factor-1 alpha) using the Q-Gene method in the Microsoft Excel-based software Visual Basic (Muller et al., 2002 (link); Simon, 2003 (link)). For each sample, three biological replicates were performed with three technical replicates per biological replicate.