Total bacteria, nitrate-reducing bacteria and cellulolytic bacteria species were quantified using SYBR Green PCR RealMaster Mix (Tiangen Biotech, co., LTD, China) on an ABI 7300 Prism real-time PCR instrument (ABI, Foster City, CA, USA). They were quantified using the following PCR program, one cycle at 95°C for 15 min (initial denaturation), 40 cycles at 95°C for 15 s (denaturation) and 60°C for 32 s (annealing). All real-time PCR assays were performed triplicate. And primers sets are shown in Supplementary Table S1 .
Sybr green pcr real master mix
Manufactured by Tiangen Biotech
Sourced in China
SYBR Green PCR Real Master Mix is a ready-to-use solution for real-time PCR amplification and detection. It contains SYBR Green I dye, DNA polymerase, and necessary reaction components.
Automatically generated - may contain errors
Lab products found in correlation
Cfx96 real time system thermal cycler, by Bio-Rad (3 mentions)
Dnase 1, by Promega (3 mentions)
Goscript reverse transcription system, by Promega (2 mentions)
Trizol reagent, by Takara Bio (2 mentions)
Rq1 dnase, by Promega (1 mentions)
Abi 7500, by Thermo Fisher Scientific (1 mentions)
Spss statistics 21, by IBM (1 mentions)
Cfx96 real time system, by Bio-Rad (1 mentions)
Trizol reagent, by Thermo Fisher Scientific (1 mentions)
E z n a plant rna kit, by Omega Bio-Tek (1 mentions)
Software 7500 ver 2, by Tiangen Biotech (1 mentions)
Abi 7500 real time pcr system, by Tiangen Biotech (1 mentions)
Superscript 3 reverse transcriptase, by Thermo Fisher Scientific (1 mentions)
Tranzol up, by Transgene (1 mentions)
Mircute plus mirna qpcr detection kit, by Tiangen Biotech (1 mentions)
Mircute plus mirna first strand cdna synthesis kit, by Tiangen Biotech (1 mentions)
11 protocols using sybr green pcr real master mix
1
Quantifying Bacterial Communities via RT-PCR
2
Quantitative Real-Time PCR of Fecal Microbiome
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Quantitative real-time PCR with specific primers and probes was used to determine the population size of thirteen major fecal bacteria. All the qPCR assays were quantified using SYBR Green PCR RealMaster Mix (Tiangen Biotech, co., LTD, China) on an ABI-7300 Prism real-time PCR instrument (ABI, Foster City, CA). The following PCR program was used: one cycle of 95 °C for 15 min (initial denaturation), 40 cycles of 95 °C for 15 s (denaturation) and 60 °C for 32 s (annealing). Following qPCR, the products of amplification were confirmed by agarose gel (1.2%) electrophoresis. To minimize variations, all real-time PCR assays were performed in triplicate. The abundance of fecal microbes was recorded and multiplied by the dilution factor to determine the total number of target microbes per gram (wet weight).
3
RNA Extraction and Real-Time PCR Analysis
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Total RNA was extracted using an RNAprep pure plant total RNA extraction kit (Kangwei, Beijing, China). DNA contamination was eliminated by adding RQ1 DNAse (Promega). Total RNA concentrations of samples were routinely determined from A260 and A280 values and integrity was checked by agarose gel electrophoresis.
RNA samples were reverse-transcribed into cDNAs according to the kit manufacturer’s instructions (Tiangen Biotech Co. Ltd, Beijing, China). The resulting cDNAs were used as templates for PCR amplification. To confirm accuracy, all PCR products of the expected size were sequenced (Sangon Biotech Co. Ltd, Shanghai, China).
Primers were designed using Primer Express 3.0 software. The PCR primer sequences are listed inTable 1 . Real-time PCR analysis was performed as follows. The cDNA samples were used as the template and mixed with 200 nmol of each primer and SYBR Green PCR Real Master Mix (Tiangen Biotech Co. Ltd, Beijing, China) for real-time PCR analysis using an ABI 7500 (Applied Biosystems, USA). To determine relative gene expression for each sample, the threshold cycle (Ct) value was normalized to actin and set relative to control samples according to the 2-ΔΔCt method.
RNA samples were reverse-transcribed into cDNAs according to the kit manufacturer’s instructions (Tiangen Biotech Co. Ltd, Beijing, China). The resulting cDNAs were used as templates for PCR amplification. To confirm accuracy, all PCR products of the expected size were sequenced (Sangon Biotech Co. Ltd, Shanghai, China).
Primers were designed using Primer Express 3.0 software. The PCR primer sequences are listed in
4
Quantitative Analysis of CircRNAs
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A set of divergent primers were designed based on the flanking sequences of head-to-tail splicing sites of circRNAs to confirm and quantify the circRNAs predicted in this study (Additional file 1 : Table S1). The primers used for the quantitative analysis of the circRNA-host genes were designed using the Primer 5.0 software and listed in Additional file 1 : Table S1. The cDNA samples were used as templates and mixed with primers and SYBR Green PCR Real Master Mix (Tiangen, China) for real-time PCR analysis using a CFX96 Real-Time System (Bio-Rad, USA). The temperature procedure was: 95 °C for 5 min followed by 40 cycles of 95 °C for 15 s, 60 °C for 15 s, and 72 °C for 15 s. TaActin was used as a reference gene to normalize the expression level of investigated genes. IBM SPSS statistics 21 software was used to determine the statistical significance of the data.
5
Profiling Root Apical Zone Gene Expression
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The meristematic, elongation and maturation zones of root tips were dissected by a shaving blade under a microscope (OLYMPUS BX-53, Japan). The whole root samples and different root apical zone samples were quick-frozen in liquid nitrogen and stored in a −80 °C freezer for RNA extraction. Total RNA of each sample was isolated using TRIzol Reagent (Invitrogen, USA) with genomic DNA removed using DNase I (Promega, USA). Each total RNA sample was used to synthesize the first strand of cDNA using the GoScript Reverse Transcription System (Promega, USA) following the manufacturer’s instructions. Primer 5.0 software (Premier, Canada) was used to design real-time PCR primers (Supplementary Table S6 ). SYBR Green PCR Real Master Mix (Tiangen, Beijing, China) was used for real-time PCR analysis on a Thermal Cycler CFX96 Real-Time System (Bio-Rad, USA) according to the instructions. The relative expression levels of target genes were computed using the 2−ΔΔCT method, with TaActin as the reference gene.
6
Methanogen Abundance Quantification by qPCR
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Total genomic DNA from 17 samples was extracted while one 2NR sample was excluded due to improper storage. A DNA extraction toolkit was adopted for DNA extraction (Tiangen Biotech Co., Beijing, China) combined with an oscillator (Precellys 24, Bertin Technology, Montigny-le-Bretonneux, France). Rotating speed of the oscillator was 5500 rpm with two circulations and 30 s per circulation.
Total methanogen abundance was quantified with an ABI 7300 Prism real-time PCR (ABI, Foster City, CA, USA) using SYBR Green PCR RealMaster Mix (Tiangen Biotech, co., LTD, China). Methanogen 16 s rRNA sequences were amplified using primers Met630F/Met803R (Met630F-GGATTAGATACCCSGGTAGT; Met803R-RGTTGARTCCAATTAAACCGCA) [19 (link)] on the following PCR program: one cycle of 95 °C for 15 min (initial denaturation), 35 cycles of 95 °C for 30 s (denaturation), 60 °C for 30 s (annealing) and 72 °C for 30 s (elongation), followed by a final step of 72 °C for 5 min. All real-time PCR assays were performed in triplicate.
Total methanogen abundance was quantified with an ABI 7300 Prism real-time PCR (ABI, Foster City, CA, USA) using SYBR Green PCR RealMaster Mix (Tiangen Biotech, co., LTD, China). Methanogen 16 s rRNA sequences were amplified using primers Met630F/Met803R (Met630F-GGATTAGATACCCSGGTAGT; Met803R-RGTTGARTCCAATTAAACCGCA) [19 (link)] on the following PCR program: one cycle of 95 °C for 15 min (initial denaturation), 35 cycles of 95 °C for 30 s (denaturation), 60 °C for 30 s (annealing) and 72 °C for 30 s (elongation), followed by a final step of 72 °C for 5 min. All real-time PCR assays were performed in triplicate.
7
Quantitative RT-PCR Gene Expression Analysis
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Six functionally important and representative genes were selected for validation of gene regulation using quantitative RT-PCR. Gene-specific primers were designed for each gene, and the ADH3 gene was used as the internal control [25 (link)]. Total RNAs of peanut leaves were extracted using E.Z.N.A. Plant RNA Kit (Omega Bio-tek, USA, R6827-01). Samples of 1 μg RNA were used to synthesize the first-strand cDNA using a reverse-transcription system (Promega, USA, A3500) according to the manufacturer’s instructions. Quantitative real-time PCR was performed on the Eppendorf Realplex (4) PCR machine (Eppendorf, Hamburg, Germany) using a reaction volume of 20 μl contained 2× SYBR Green PCR RealMaster Mix (TianGen, FP202-02), forward and reverse primers, and cDNA template (diluted 1:40). All samples were run in duplicate. The real-time PCR reaction was run for one cycle (94°C for 3 min) followed by 40 cycles (94°C for 15 s, 59°C for 20 s, 68°C for 40 s) and fluorescence emissions were measured after each of the repetitive cycles. The results were obtained based on the average of three parallel experiments.
8
Quantification of Gene Expression in Oriental Melon
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The total RNA was isolated with TRIzol Reagent (Takara, Japan). DNase I (Promega, USA) was used to remove genomic DNA. The total RNA extracted from fruit was used to generate cDNA samples via random priming with Superscript III reverse transcriptase (Invitrogen, Thermo Fisher Scientific, USA).
The cDNA samples were used as templates and were mixed with 10 μM of each primer and SYBR Green PCR Real Master Mix (Tiangen, Beijing, China) for real-time PCR analysis using the ABI 7500 Real Time PCR System and Software 7500 ver. 2.0.3 (Applied Biosystems, USA) as described in the manufacturer’s instructions. The temperature procedure was: 95 °C for 15 min; and 40 cycles of 95 °C for 30 s, 57 °C for 30 s, and 68 °C for 1 min. The fluorescence signal was collected during the elongation at 68 °C of every cycle. The oriental melon 18S rRNA was used as an internal control to normalize small differences in the template amounts. The LOX/18SrRNA, AAT/18SrRNA, SS/18SrRNA and SPS/18SrRNA ratios for all samples were related to the ratio for 5 DAA, which was set to 1. The primers used for real-time qPCR are listed in Additional file1 .
The cDNA samples were used as templates and were mixed with 10 μM of each primer and SYBR Green PCR Real Master Mix (Tiangen, Beijing, China) for real-time PCR analysis using the ABI 7500 Real Time PCR System and Software 7500 ver. 2.0.3 (Applied Biosystems, USA) as described in the manufacturer’s instructions. The temperature procedure was: 95 °C for 15 min; and 40 cycles of 95 °C for 30 s, 57 °C for 30 s, and 68 °C for 1 min. The fluorescence signal was collected during the elongation at 68 °C of every cycle. The oriental melon 18S rRNA was used as an internal control to normalize small differences in the template amounts. The LOX/18SrRNA, AAT/18SrRNA, SS/18SrRNA and SPS/18SrRNA ratios for all samples were related to the ratio for 5 DAA, which was set to 1. The primers used for real-time qPCR are listed in Additional file
9
Transcriptional Analysis of C. oleifera Buds
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Total RNA was extracted from the buds of C. oleifera using the TRIZOL reagent (Takara, Japan) (Du et al., 2022 (link)). cDNA was synthesized by reverse transcription, and the cDNA samples were mixed with SYBR Green PCR Real Master Mix (Tiangen, China) and 10 μmol/L of each primer. Applied Biosystems 9700 (ABI, USA) was used to conduct PCR. The program settings were as follows: heating for 15 min at 95°C, followed by 42 cycles of 32 s at 95°C, 30 s at 59°C, and 48 s at 67°C. The Primer Quest online software was used to design the qRT-PCR primers (Table S2
10
Quantitative Real-Time PCR Analysis
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Total RNA was extracted using TranZol Up (Transgen, USA). DNase I (Promega, USA) was used to remove genomic DNA. Total RNA extracted from roots was used to generate cDNA samples via random priming with the GoScript Reverse Transcription System (Promega, USA). Six DAPs (including two nitrogen transporters and four randomly selected proteins) were selected for RNA level examination to verify the iTRAQ data and investigate the correlation of protein species abundance with their corresponding mRNA level. The cDNA samples were used as templates and mixed with primers and SYBR Green PCR Real Master Mix (Tiangen, China) for real-time PCR analysis using Thermal Cycler CFX96 Real-Time System (Bio-Rad, USA). The temperature procedure was: 95°C for 15 min followed by 40 cycles of 95°C for 15 s, 60°C for 15 s, and 72°C for 15 s. TaActin was used as a reference gene to normalize the expression level of target genes. 2−ΔΔCT method was employed to calculate the relative gene expression level. Three biological replicates were performed. The primers used for real-time PCR were designed using Primer 5.0 software and are listed in Table S1 .
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