TRIzol (15596,018, Ambion, USA) was used to isolate total RNAs from the cells, tissues, and exosomes. Tiagen (KG203, Beijing, China) provided the DNA extraction kit for cell genomic DNA extraction. The cDNA Synthesis SuperMix (11120ES60, Yeasen) was used to reverse transcript circRNA and mRNA into cDNA. The miRNA cDNA Synthesis Kit (R601, EnzyArtisan Biotech, China) was used to reverse transcript miRNA into cDNA. The QPCR SYBR Green Mix (11201ES08, Yeasen, China) was used for conducting qPCR. Electrophoresis was performed on 2% agarose gel after PCR. For qRT-PCR, the PCR products were quantified on the MX3000P (Agilent, USA). The primers were synthesized by the EnzyArtisan Biotech (China), and the 2-ΔΔCt method was used for calculation [38 (link)]. The primer sequences are listed in Table S3.
Cdna synthesis supermix
Manufactured by Yeasen
Sourced in China
The CDNA Synthesis SuperMix is a reagent kit designed for the reverse transcription of RNA into cDNA. It contains all the necessary components for the efficient conversion of RNA to cDNA in a single, ready-to-use mix.
Automatically generated - may contain errors
Lab products found in correlation
Sybr green master mix, by Yeasen (5 mentions)
Qpcr sybr green master mix, by Yeasen (4 mentions)
Trizol, by Thermo Fisher Scientific (3 mentions)
Trieasy reagent, by Yeasen (2 mentions)
Trizol reagent, by Yeasen (2 mentions)
Transcript one step gdna removal, by Yeasen (2 mentions)
Trizol reagent, by Thermo Fisher Scientific (2 mentions)
Random primer, by Promega (2 mentions)
Sybr green qpcr mix, by Yeasen (1 mentions)
Mx3000p, by Agilent Technologies (1 mentions)
7500 sequence detection system, by Thermo Fisher Scientific (1 mentions)
Hieff qpcr sybr green master mix, by Yeasen (1 mentions)
Steponeplus, by Thermo Fisher Scientific (1 mentions)
Abi 7500 real time pcr system, by Thermo Fisher Scientific (1 mentions)
Sybr green qpcr mix, by MedChemExpress (1 mentions)
Quantstudio 5, by Thermo Fisher Scientific (1 mentions)
Universal sybr green fast qpcr mix, by ABclonal (1 mentions)
Total rna extraction kit, by Qiagen (1 mentions)
Lightcycler 480, by Yeasen (1 mentions)
Mx3005p qpcr system, by Agilent Technologies (1 mentions)
22 protocols using cdna synthesis supermix
1
Total RNA, DNA, and cDNA Extraction and Quantification Protocol
2
Quantifying mRNA Expression in Cells
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The mRNA expression level was detected by qRT-PCR with qPCR SYBR Green Master Mix (Yeasen Biotechnology, Shanghai, China) in a 7500-sequence detection system (Applied Biosystems, Waltham, MA, USA) according to the manufacturer’s instruction. Total RNA was extracted from cells using TRIeasy reagent (Yeasen Biotechnology, Shanghai, China). The mRNA was reversely transcribed into cDNA using the cDNA Synthesis SuperMix (Yeasen Biotechnology, Shanghai, China). Three duplicates were designed for each sample, and the fold change was measured using the 2−ΔΔCT method, with GAPDH as an internal reference. The primer sequences synthesized by Rui Biotech (Beijing, China) were as follows: MDK, 5′-CGCGGTCGCCAAAAAGAAAG-3′ (forward), 5′-TACTTGCAGTCGGCTCCAAAC-3′ (reverse); GAPDH, 5′-AATCCCATCACCATCTTCCA-3′ (forward), 5′-TGGACTCCACGACGTACTCA-3′ (reverse); and Ki67, 5′-AGAAGAAGTGGTGCTTCGGAA-3′ (forward), 5′-AGTTTGCGTGGCCTGTACTAA-3′ (reverse).
3
RNA Extraction and qPCR Analysis
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Total RNA was extracted and reverse transcribed using TRIzol reagent and cDNA Synthesis SuperMix (Yeasen Biotech, China). Real-time polymerase chain reaction was carried out in Applied Biosystems QuantStudio 3 with Hieff qPCR SYBR Green master mix (Yeasen Biotech, China) and gene-specific primers.
4
Efficient lncRNA Expression Quantification
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Transcript One-Step gDNA Removal (YEASEN, shanghai, China) and cDNA Synthesis SuperMix (YEASEN, shanghai, China) were employed during the reverse transcription process according to the manufacturer’s instructions. cDNA was amplified with SYBR q-PCR Master Mix (EnzyArtisan, shanghai, China) in StepOnePlus (Applied Biosystems) equipment. The 2-ΔΔCt method was used to calculate the relative lncRNA expression, and each hole was repeated three times to ensure quantitative accuracy. The sequences of primers used for qRT-PCR are listed in Table S2 , and GAPDH was used as an internal reference gene.
5
Quantifying Tmc1 Transcript Levels in Mouse Cochleae
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Cochleae from wild-type or heterozygous mice were dissected. Total mRNA was extracted from the organs of Corti by TRIzol (Invitrogen) and mRNA was reverse-transcribed using a cDNA synthesis supermix (YEASEN) according to the manufacturer’s protocol. One microliter of RT product was added to RT-qPCR SYBR (YEASEN) for subsequent RT-qPCR with the following steps: 95 °C for 5 min and 40 cycles of 95 °C for 10 s and 60 °C for 35 s. Primers were designed to detect the total Tmc1 expression level: q-TMC1-F2 and q-TMC1-R2. To detect the mutant transcript expression, the forward primer, q-TMC1-F4 was designed with a 3′ end ‘A’ that specifically bound to the mutant sequence; to detect the wild-type transcript expression, the forward primer, q-TMC1-F5 was designed with a 3′ end ‘T’ that specifically bound to the wild-type sequence, and q-TMC1-R2 was used as reverse primer.
6
Quantitative Analysis of SFRP2 Expression
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Total RNA was extracted from JEG-3 cells using the RNA-iSo PluS kit (cat. no. 9109; Takara Biotechnology Co., Ltd.). RNA was reverse-transcribed using cDNA Synthesis SuperMix (Shanghai Yeasen Biotechnology Co., Ltd.; cat. no. 11119ES60). RT was performed as follows: Initial hold at 25°C for 5 min, followed by 42°C for 30 min and final elongation at 85°C for 5 min. Synthesized cDNA was subjected to qPCR amplification using SYBR Green qPCR Mix (MedChemExpress; cat. no. HY-K0501A) and analyzed on ABI 7500 Real-Time PCR system (Thermo Fisher Scientific, Inc.). Each 20 µl qPCR reaction mixture included 1.0 cDNA, 10.0 SYBR Premix Ex Taq (2X) and 0.4 µl each primer (10 µM) and was completed with double-distilled water. Amplifications followed a two-step cycling protocol as follows: Initial denaturation at 95°C for 30 sec, followed by 40 cycles of 10 sec at 95°C and 30 sec at 60°C. The final step comprised the melting curve analysis. To normalize data, SFRP2 expression was compared with housekeeping gene GAPDH using the 2−ΔΔCq method (27 (link)). The specific primer sequences were as follows: GAPDH forward, 5′-CCAGGTGGTCTCCTCTGA-3′ and reverse, 5′-GCTGTAGCCAAATTCGTTG-3′ and SFRP2 forward, 5′-CACCGAGGAAGCTCCAAAG-3′ and reverse, 5′-CTTTCGGACACACCGTTCAG-3′.
7
Quantitative Analysis of Ovarian Gene Expression
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Total RNA was extracted from the ovaries using the MolPure® Cell/Tissue Total RNA Kit (19221ES50) following the manufacturer’s instructions. The first-strand cDNA was generated using cDNA Synthesis SuperMix (YEASEN). Relative quantitative analysis was performed using 2X Universal SYBR Green Fast qPCR Mix (Abclonal, China, RK21203) on a QuantStudio 5 instrument (Applied Biosystems, Carlsbad, CA, USA). The PCR cycling parameters were as follows: an initial denaturation at 95 °C for 10 min, followed by 40 cycles of denaturation at 95 °C for 15 s, annealing and extension at 60 °C for 60 s, and a final melting curve analysis with denaturation at 95 °C for 15 s and annealing at 60 °C for 1 min; Gapdh was used to normalize gene expression levels. The relative transcript abundance was analyzed via the 2−ΔΔCT method. Primers used in this study are shown in Table S1 .
8
Quantifying lncRNA Expression in Colon Cancer
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Eleven pairs of colon cancer and adjacent tissue samples were collected from the Second Affiliated Hospital of Zhejiang University School of Medicine. Total cellular and tissue RNA was extracted using a total RNA extraction kit (73404, Qiagen) according to standard protocol. The RNA was used to synthesize complementary DNA (cDNA) with a cDNA Synthesis SuperMix (11141ES60, Yeasen). The cDNA was used as a template and lncRNA expression was quantified with the Roche LightCycler 480 using SYBR Green Master Mix (11198ES25, Yeasen). β-actin was used as an endogenous control. Primers were synthesized by Sangon Biotech (Sangon, China). The sequences are listed in Table 1
9
Quantitative RT-PCR from Mouse Tissues
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RNA was extracted from the dorsal root ganglion and plantar tissues of mice using the Eastep® Super Total RNA Kit. The extracted RNA was then subjected to reverse transcription using the cDNA Synthesis SuperMix (YEASEN). Subsequently, the resulting cDNA was amplified using the precision Agilent Mx 3005p qPCR system. The obtained data was analyzed using the cycle threshold method. The primer sequences used in the amplification process are provided in Table 1 .
10
Quantification of Cytokine Transcripts in PBMCs
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Total RNA was extracted from PBMCs using TRIzol reagent (Ambion North America, USA) then RNA concentrations were determined via spectrophotometry. Next, 1 μg of RNA was reverse transcribed with cDNA Synthesis SuperMix (Yeasen Biotech Co., Ltd., Shanghai, China) following the manufacturer’s instructions. IL-10, TGF-β, and IL-4 mRNA levels were determined via real-time PCR using SYBR Green Master Mix (Yeasen Biotech, Shanghai, China). Primers were synthesised by a commercial primer synthesis company as follows: human IL-10, sense (5ʹ-TCAAGGCGCATGTGAACTCC-3ʹ) and antisense (5ʹ- GATGTCAAACTCACTCATGGCT-3ʹ); human TGF-β, sense (5ʹ-GCGGTACCTGAACCCGTGTT-3ʹ) and antisense (5ʹ-GTCAATGTACAGCTGCCGCAC-3ʹ); human IL-4, sense (5ʹ-CCAACTGCTTCCCCCTCTG-3ʹ) and antisense (5ʹ-TCTGTTACGGTCAACTCGGTG-3ʹ); and human glyceraldehyde-3-phosphate dehydrogenase (GAPDH), sense (5ʹ-GGAGCGAGATCCCTCCAAAAT-3ʹ) and antisense (5ʹ- GGCTGTTGTCATACTTCTCATGG-3ʹ).17 (link),18 (link) For mRNA analysis, RT of total RNA was performed using random primers (Promega); analysis of GAPDH mRNA was conducted in a separate PCR reaction for each sample as a loading control for use in normalising PCR results based on a constant amount of template cDNA across samples. Ct values were converted to fold changes in expression (2-ΔΔCt values) following normalisation based on housekeeping gene levels.
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