Total RNA was extracted and cDNA was synthesized was performed according to the instructions for the plant total RNA extraction kit (Aidlab, Beijing, China) and the Fastking RT Kit (TIANGEN, Beijing, China). The RT-qPCR analyses were performed using SYBR Green qPCR Mix (TIANGEN) on an iQ5 Multicolor Real-Time PCR detection system (Bio-Rad Laboratories, Hercules, CA, USA). The primers used for RT-qPCR are shown in Supplemental Table S1 and Arabidopsis ACTIN gene (AT2G37620) was used as the internal control. The PCR conditions were 94 °C for 3 min, 40 cycles at 94 °C for 10 s, 55 °C for 20 s, 72 °C for 20 s, and 60 °C for 30 s, and 72 °C for 1 min. All reactions were run in triplicate for each sample. Data were analyzed using the iQ5 (Bio-Rad) software (Bio-Rad Laboratories, Hercules, CA, USA), and the difference in gene expression was calculated using the 2-ΔΔCt method. Data are means (±SD) of three replicates (Supplemental Data Set S1) .
Sybr green qpcr mix
Manufactured by Tiangen Biotech
Sourced in China
SYBR Green qPCR Mix is a ready-to-use solution for quantitative real-time PCR (qPCR) analysis. It contains SYBR Green I dye, Taq DNA polymerase, dNTPs, and necessary buffers and additives for efficient DNA amplification and detection.
Automatically generated - may contain errors
Lab products found in correlation
Iq5 multicolor real time pcr detection system, by Bio-Rad (2 mentions)
Cfx96, by Bio-Rad (2 mentions)
Fastking rt kit, by Tiangen Biotech (1 mentions)
Plant total rna extraction kit, by Aidlab (1 mentions)
Quantitect reverse transcription kit, by Qiagen (1 mentions)
Cdna reverse transcription kit, by Takara Bio (1 mentions)
E z n a total rna kit 1, by Omega Bio-Tek (1 mentions)
Quantscript reverse transcriptase kit, by Tiangen Biotech (1 mentions)
Lightcycler 96 system, by Roche (1 mentions)
6 protocols using sybr green qpcr mix
1
Plant RNA Extraction and RT-qPCR Analysis
2
Quantifying Mature miRNA Expression
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To validate the expression of mature miRNAs, miRNA expression was quantified in individual samples using a Bulge-Loop reverse-transcription quantitative polymerase chain reaction (RT-qPCR) assay (RiboBio, Guangzhou, China). One reverse transcription primer and a pair of quantitative PCR primers were designed for each of miRNAs including novel_138, oar-miR-1185-5p, novel_469, oar-miR-323c, novel_96, oar-miR-150, oar-miR-29a, and novel_459. Reverse transcription of total miRNA was conducted using 1 μg total RNA per sample and the QuantiTect Reverse Transcription Kit (Qiagen). Real-time quantitative-PCR was carried out using SYBR Green qPCR mix (Tiangen, Beijing, China), with U6 small nuclear RNA as an internal reference for normalization. Quantitative-PCR reactions were performed using a BioRad CFX96 (BioRad, CA, USA). Relative miRNA expression was calculated using the standard curve-based method for relative real-time PCR, which has been previously described [25 (link)].
3
qPCR Assay for Klebsiella pneumoniae rmpA2
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A pair of specific primers (forward: 5’-TGATTATGACATCTAAGTCTACATGCAAGG-3’; reverse: 5’-TTTACATCTGTGACACGATAGTGTTTTCTC-3’) targeting the virulence gene rmpA2 of Klebsiella pneumoniae was used for qPCR. The qPCR reaction mixture contained 10 µl of 2 × SYBR Green qPCR Mix (Tiangen Biotech Co. Ltd), 0.4 µM of each primer, 1 µl of the template, and 8.2 µl of distilled water. The cycling program was 95°C for 30 s, followed by 40 cycles of 95°C for 10 s, then 60°C for 30 s. The melting curve analysis was set as default. Cycle threshold (Ct) values less than 32 were considered as positive.
4
Quantifying PtCP5 Gene Expression in P. trichoderma
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To analyze the expression levels of PtCP5, RNA was extracted from the leaf, stem and root of the tissue culture of a P. trichoderma plant aged three months. The RT-qPCR analyses were performed using SYBR Green qPCR Mix (TIANGEN, Beijing, China) on an iQ5 Multicolor Real-Time PCR detection system (Bio-Rad Laboratories, Hercules, CA, USA). The PCR conditions were 94 °C for 3 min, 40 cycles at 94 °C for 10 s, 55 °C for 20 s, 72 °C for 20 s, 60 °C for 30 s and 72 °C for 1 min. Data were analyzed using iQ5 (Bio-Rad) software, and differences in gene expression were calculated using the 2−ΔΔCt method. The β-actin, acting as an internal control, was used to quantify the relative expression levels of genes in samples. There were three technical replicates and three biological replicates.
5
Quantitative Gene Expression Analysis in Caco-2 Cells
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The Caco‐2 cells and colon tissues were removed from −80 °C, and RNA was extracted using an E.Z.N.A Total RNA Kit I (Omega Biotek) according to the protocol. Reverse transcription was performed using a cDNA Reverse Transcription Kit (Takara). qPCR was performed with SYBR green qPCR Mix (TIANGEN) on a CFX96 (BiO‐RAD). The cycling conditions were 95 °C for 15 min, followed by 40 cycles of 95 °C for 10 s, 58 °C for 30 s, and 72 °C for 30 s. The primers used for amplification were described.[42 ]
6
Quantitative Analysis of Rat FGL2 Expression
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The total RNA of recipient livers and spleens were isolated using Qiagen RNA Minikit (Tiangen Biotech, Beijing, China). The RNA quantity and quality were detected using the ND-1000 Spectrophotometer (NanodropTM, Rockland, DE, USA). The first-strand complement DNA (cDNA) was synthesized by a Quantscript Reverse Transcriptase Kit (Tiangen Biotech, Beijing, China). The quantitative reverse transcription polymerase chain reaction qRT-PCR was performed by using SYBR green qPCR mix (Tiangen Biotech, Beijing, China) according to manufacturer’s instructions. The optimal qRT-PCR efficacy was achieved using a cycling profile including a denaturation at 95 °C for 15 min, followed by 40 cycles of 10 s at 95 °C, 20 s at 60 °C, and 30 s at 72 °C. Anti-glyceraldehyde 3-phosphate dehydrogenase (GAPDH) antibody was used as the internal reference. The qRT-PCR test was executed in a fluorescence ration PCR instrument (LightCycler® 96 System, Roche Life Science, Penzberg, Germany). All qRT-PCR experiments were performed in duplicate. The sequence of primer pairs for rat FGL2 was 5'-ATGGGAGCACCAACTTCACC-3' (forward) and 5'-TCGTACACGGCGTAAAGTGT-3' (reverse), GAPDH was 5'-GGCAAGTTCAACGGCACAG-3' (forward) and 5'-CGCCAGTAGACTCCACGACAT-3' (reverse).
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