qRT-PCR analyses were performed in 96-well blocks with a BIO-RAD CFX96 Real-Time PCR system (Bio-Rad, Hercules, California, USA) using NovoStart SYBR qPCR Supermix Plus (Novoprotein, China) in a 10-μL volume, containing 5 μL 2× NovoStart SYBR qPCR Supermix Plus, 1 μL diluted cDNA, 0.5 µL of forward primer (10 µmol/L), 0.5 µL of reverse primer (10 µmol/L), and 3 µL of ddH2O. The cycling conditions were as recommended by the manufacturer: 1 min at 95 °C, followed by 39 cycles of 95 °C for 5 s, 60 °C for 30 s, and 95 °C for 5 s. To confirm the specificity of the primers, melting curves were included after amplification. At the end of the reaction process, the melting curve was derived by heating the amplicon from 65 to 95 °C. All qRT-PCR analyses were run in technical quadruplicates and biological triplicates.
Novostart sybr qpcr supermix plus
Manufactured by Novoprotein
Sourced in China, United States
NovoStart® SYBR qPCR SuperMix Plus is a ready-to-use solution for quantitative real-time PCR (qPCR) experiments using SYBR Green technology. It contains all the necessary components for efficient and sensitive qPCR reactions, including a high-performance DNA polymerase, SYBR Green I dye, and optimized buffer system.
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Lab products found in correlation
Trizol reagent, by Thermo Fisher Scientific (24 mentions)
Trizol, by Thermo Fisher Scientific (20 mentions)
Nanodrop, by Thermo Fisher Scientific (8 mentions)
Nanodrop 2000, by Thermo Fisher Scientific (8 mentions)
Rnaiso plus, by Takara Bio (7 mentions)
Novoscript plus all in one 1st strand cdna synthesis supermix gdna purge, by Novoprotein (7 mentions)
Primescript rt reagent kit with gdna eraser, by Takara Bio (7 mentions)
Steponeplus real time pcr system, by Thermo Fisher Scientific (7 mentions)
Novoscript plus all in one 1st strand cdna synthesis supermix, by Novoprotein (6 mentions)
Cfx connect real time system, by Bio-Rad (4 mentions)
Lightcycler 96 system, by Roche (4 mentions)
Rneasy mini kit, by Qiagen (4 mentions)
Nanodrop one spectrophotometer, by Thermo Fisher Scientific (4 mentions)
Cfx96 real time system, by Bio-Rad (4 mentions)
High capacity cdna reverse transcription kit, by Thermo Fisher Scientific (4 mentions)
Cfx96 real time pcr system, by Bio-Rad (3 mentions)
Hiscript 2 q rt kit, by Vazyme (3 mentions)
Cfx96 instrument, by Bio-Rad (3 mentions)
Novoscript plus all in on strand cdna synthesis supermix, by Novoprotein (3 mentions)
Cfx96 touch real time pcr detection system, by Bio-Rad (3 mentions)
117 protocols using novostart sybr qpcr supermix plus
1
Quantitative Real-Time PCR Protocol
2
Comparative TSSK1B Expression Analysis in Yak and Cattle-Yak
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The relative mRNA expression of TSSK1B in each tissue sample of yak and cattle–yak was detected by RT-qPCR, following the previous study [20 (link)]. The primers of TSSK1B for RT-qPCR were designed, as shown in Table 1 . Each amplification system (20 μL) included 1 μL of forward and reverse primers, 10 μL of 2×NovoStart®SYBR qPCR SuperMix Plus (Novoprotein, Suzhou, China), and 1 μL of diluted cDNA supplied with 7 μL RNase free water up to 20 μL. The CFX96TM system (Bio-Rad, Hercules, CA, USA) was used with the following program steps: 95 °C for 1 min, 40 cycles of 95 °C for 20 s, and 60 °C for 1 min, followed by dissociation curve and cool down. The relative fold change of genes was calculated by 2–∆∆Ct method, and glyceraldehyde-3-phosphate dehydrogenase (Gapdh) was used as the housekeeping gene for data normalization. Each sample analysis was repeated independently in triplicate. For easy comparison of the relative expression of TSSK1B in various organs, the expression in heart was set to 1 (control group). The TSSK1B expression of adult yak testis was also set as the control group when contrasting the differential expression in yak testes of different ages and the expression in adult cattle–yak testes.
3
Nile Tilapia Transcriptome Analysis
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Total RNA was extracted from spleen, head kidney, trunk kidney, liver, intestine, gill and peripheral blood of Nile Tilapia using TRIzol reagent (Invitrogen). The extracted RNA was synthesized into cDNA using reverse transcription kit (Novoprotein). The synthesized cDNA was diluted at 1:30 as the template and β-actin was used as the internal reference gene. The relative mRNA levels of target genes were detected by 2 × NovoStart SYBR qPCR SuperMix Plus (Novoprotein), and the relative expression levels of target genes were calculated by 2−△△CT method. The gene-specific primers used in this study are listed in Table S2.
4
Quantitative RT-PCR Analysis of Plant Transcripts
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Total RNA was extracted using the EASY spin plant RNA extraction kit (Aidlab Biotech, China). Approximately 1 μg RNA was transcribed into first-strand cDNA using the NovoScript Plus All-in-one First Strand cDNA Synthesis SuperMix (gDNA Purge, Novoprotein, China). The real-time quantitative PCR (RT-qPCR) assays were performed on a CFX Connect Real-Time System (Bio-Rad Laboratories) with 2×NovoStart SYBR qPCR SuperMix plus (Novoprotein, China). GhHis3 (Zhang et al., 2011 ; Wan et al., 2016 (link); Wu et al., 2018 (link)) and GhUbiquitin (Walford et al., 2011 (link); Wu et al., 2018 (link)) served as internal references. Gene specific primers used for RT-qPCR are listed in Supplementary Table S2 . The expression data were calculated with the ΔΔCt method. For the RT-qPCR analysis, three individual biological replicates with two technical replicates for each gene were used. Mean values and standard errors were calculated using the data from the three replicates. The compliance of the RT-qPCR experiments with the Minimum Information for Publication of Quantitative Real-Time PCR Experiments (MIQE) is shown in a MIQE checklist (Supplementary Table S3 ).
5
Quantitative Analysis of Gene Expression
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The gene relative expression in the sample’s cDNA was detected by RT-qPCR using a NovoStart® SYBR qPCR SuperMix Plus kit (Novoprotein, Suzhou, China). It comprised 1.0 μL of template, 5.0 μL of 2× NovoStart® SYBR qPCR SuperMix Plus (Novoprotein, China), 0.5 μL of each primer (10 μM) and 3.0 μL of double-distilled water (ddH2O). The qPCR reaction was carried out in the LightCycler® 96 system (Roche, Basel, Switzerland). The thermal cycling program consisted of pre-denaturation at 95 °C for 5 min, 40 cycles at 95 °C for 15 s and 60 °C for 30 s. A melt curve analysis, performed at the end of the PCR cycles, displayed a single sharp peak from 80 to 85 °C. After obtaining the Cq value of the sample, the relative expression level was calculated by the 2−ΔΔCT method. The statistical difference between the three biological replicates was calculated via one-way analysis of variance. GraphPad Prism was used to plot the graph. Glyceraldehyde-3-phosphate dehydrogenase (BmGAPDH) was used as an internal reference gene, and late expression factor 3 (lef3) was used to detect AcMNPV. The primers are shown in Table 2 .
6
Quantifying Gene Expression in Silkworm Tissues
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The expression levels of target genes in JS and L10 were detected by RT-qPCR. The cDNA synthesized by total RNA of the midgut, silk gland, and fat body at the third day of fifth instar larval stage in JS and L10 was used as the template. The program of PCR was incubation at 95°C for 5 min, 40 cycles of 95°C for 5 s, and 60°C for 31 s, and a final dissociation. The RT-qPCR was performed in a 10 μL reaction volume that contained 1 μL template, 5 μL 2×NovoStart® SYBR qPCR SuperMix Plus (Novoprotein), 0.2 μL ROX Reference Dye II (ROX II; Novoprotein), and 0.5 μL specific primers (10 μM). Bombyx mori glyceraldehyde-3-phosphate dehydrogenase (Bm GAPDH) was used as the reference gene. The relative expression level of our target gene was calculated by using the cycle threshold value (Ct) by the method of 2-ΔCt, where ΔCt = Cttargetgene−CtBmGAPDH. The comparisons between expressions of these genes were performed with a two-tailed t-test: * P ≤ 0.05; ** P ≤ 0.01; *** P ≤ 0.001. The primers are listed in Table 1 .
7
RT-qPCR protocol for HSF2 expression
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The related primers for RT-qPCR were designed with online software and shown in Table 1 . The relative mRNA expression of the HSF2 gene was normalized to the mean abundance of the endogenous control gene beta-actin (β-actin). The amplification system (10 µL) included 5 µL 2× NovoStart®® SYBR qPCR SuperMix Plus (Novoprotein, Suzhou, China), 1.5 µL cDNA, 0.4 µL forward and reverse primers, and RNase-free water 2.7 µL. Next, the CFX96TM system (Bio-Rad, Hercules, CA, USA) was used for detecting the expression of HSF2, as in our previous study [29 (link)]. The relative expression level against β-actin was analyzed with the help of the 2−ΔΔCt [30 (link)] method. The detections were repeated at least three times. In order to make the analysis more convenient, the expression of the spleen was treated as 1 and set as the control group in tissue differential expression analysis. Similarly, the fetal cattle-yak group was defined as 1 when comparing HSF2 expression in different species (cattle, yak, and cattle-yak).
8
Quantifying Gene Expression in Nile Tilapia
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TRIzol reagent (Invitrogen) was used to extract the total RNA from spleen lymphocytes or the indicated tissues of Nile tilapia according to the manufacturer's instructions. The RNA was reverse-transcribed to cDNA using Plus All-in-one 1st Strand cDNA Synthesis SuperMix (Novoprotein). Using diluted cDNA as template, qPCR was performed with 2 × NovoStart SYBR qPCR SuperMix Plus (Novoprotein) on the QuantStudio5 Q5 (Applied Biosystems). β-actin was chosen as internal control, and the relative mRNA levels of target genes were calculated by the 2−△△CT method. The used primers for qPCR were listed in the Table S2 .
9
Quantitative Analysis of SNRPA Expression
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The total RNA was extracted from the cells using RNAiso Plus (TaKaRa, 9109, China) and then reverse transcribed into cDNA for subsequent PCR assay using gDNA Purge (Novoprotein, E047-01A, China), according to the manufacturer’s instruction. Then, NovoStart® SYBR qPCR SuperMix Plus (Novoprotein, E096-01B, China) and 7300 Real-Time PCR System (Applied Biosystems, USA) were used to perform the RT-qPCR. GAPDH was used as the internal control for SNRPA. The 2-ΔΔCt method was used to calculate the mRNA expression of SNRPA. The primer pairs of SNRPA and GAPDH were designed as following: SNRPA Forward: 5’-CAAACCTATGCGTATCCAGT-3’, Reverse: 5′‐GGATTCTCAGAAAGAGGCTG-3’ and GAPDH Forward:5’-ATGGGGAAGGTGAAGGTCG-3’, Reverse: 5’-TCGGGGTCATTGATGGCAACAATA-3’.
10
Quantitative Gene Expression Analysis
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Total RNA was extracted using the TRIzol® reagent (Invitrogen). The cDNA was prepared with NovoScript® Plus All-in-one 1st Strand cDNA Synthesis SuperMix (gDNA Purge). (Novoprotein, Shanghai, China). Gene transcripts were quantitated using the QuantStudio Three Real-Time PCR System (Thermo Fisher) using the NovoStart® SYBR qPCR SuperMix Plus (Novoprotein), with GAPDH as an internal control. The primers sequence used during our quantitative real-time polymerase chain reaction (qRT-PCR) test is shown in Supplementary Table 1 .
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