The RNA was extracted from samples using TRIzol, and Vazyme HiScript II Q RT SuperMix (Vazyme, China) was used for reverse transcription. The RT-PCR reactions were carried out as follows: 5 min at 37°C, 5 sec at 85°C, and final reduction to 4°C. qPCR was carried out using SYBR Premix Ex Taq II (Vazyme, China). The primers used (Sangon Biotech, China) are listed in Table 2 . The GAPDH was utilized as an internal reference to calculate the relative mRNA expression of target genes by the 2−(ΔΔCt) method.
Sybr premix ex taq 2
Manufactured by Vazyme
Sourced in China
SYBR Premix Ex Taq II is a ready-to-use master mix for real-time PCR and qPCR applications. It contains a hot-start Taq DNA polymerase, SYBR Green I dye, and optimized buffer components.
Automatically generated - may contain errors
Lab products found in correlation
Trizol reagent, by Thermo Fisher Scientific (3 mentions)
Primescript rt reagent kit with gdna eraser, by Vazyme (2 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (2 mentions)
Hiscript 2 q rt supermix, by Vazyme (1 mentions)
Rnaiso plus, by Vazyme (1 mentions)
Spark script 2 rt plus kit, by Sparkjade (1 mentions)
Sparkeasy improved tissue cell rna kit, by Sparkjade (1 mentions)
Trizol reagent, by Magen Biotechnology Co (1 mentions)
Primescript rt reagent kit, by Vazyme (1 mentions)
Abi q6 fast real time pcr system, by Thermo Fisher Scientific (1 mentions)
Prime script rt master mix, by Transgene (1 mentions)
Applied biosystems 7500 real time pcr system, by Thermo Fisher Scientific (1 mentions)
Nucleospin rna kit, by Takara Bio (1 mentions)
Steponeplus system, by Thermo Fisher Scientific (1 mentions)
Lightcycle 96 real time pcr system, by Roche (1 mentions)
M mlv reverse transcriptase, by Promega (1 mentions)
Primescript rt reagent kit with gdna eraser, by Takara Bio (1 mentions)
Primesript rt reagent kit, by Tiangen Biotech (1 mentions)
Rnaiso plus reagent, by Takara Bio (1 mentions)
Lipopolysaccharide, by Merck Group (1 mentions)
12 protocols using sybr premix ex taq 2
1
RNA Extraction and qPCR Analysis
2
Ovarian Gene Expression Analysis in Mice
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total RNA of ovaries of 3-week-old wt (n=5) and tg (n=5) female mice was extracted using RNAiso Plus reagent (Vazyme, Nanjing, China) and was synthesized to cDNA by reverse transcription PCR (RT-PCR) PrimeSript™ RT reagent Kit (Vazyme). Then, cDNA was used to detect the gene expression level by using qRT-PCR analysis as follows: 10 μL SYBR® Premix Ex Taq II (Vazyme), 1 μL cDNA, 10 μmol/L PCR Forward/Reverse Primer, and added RNase-free water to a total volume of 20 μL. Each sample was run in triplicate. The relative transcriptional level of the target gene was normalized to the 36B4 expression level using the method of 2−ΔΔCt (Livak and Schmittgen, 2001 (link)). The specific primers of the Pai gene used in this study were: forward: 5-tgacgagcgggaatacttta-3; reverse: 5-gagggtcatcagcccatcta-3. The specific primers of the 36B4 gene (housekeeping gene) used in this study were: forward: 5-cactggtctaggacccgagaag-3; reverse: 5-ggtgcctctggagattttcg-3.
3
Tissue/Cell RNA Extraction and RT-qPCR Assay
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
SPARKeasy Improved Tissue/cell RNA kit (AC0202; Sparkjade, China) was used to extract total RNA from testes. Equal amounts of RNA (300μg/mL) were reverse transcribed to cDNA using the SPARK script II RT Plus Kit (AG0401; Sparkjade) and RT-qPCR was carried out on a CFX96 Real-Time System instrument, including SYBR Premix Ex Taq™ II (Q711-02; Vazyme, China), RNase-free-H2O and primers (Table S4 ).
4
Quantitative Analysis of Inflammatory Markers in Colon Cancer
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total RNA was extracted from colon cancer lines using Trizol reagent (Magen, R4801-02, Shanghai, China) and 1 µg of total RNA was reverse transcribed using the PrimeScript RT reagent kit (Vazyme, Nanjing, China) to detect relevant mRNAs. Real-time fluorescence quantitative PCR was performed using SYBR Premix Ex Taq II (Vazyme, Nanjing, China) at 95 °C for 30 s, followed by 40 cycles of 95 °C for 5 s, 55 °C for 30 s, and 72 °C for 30 s. The primer sequences used for PCR are shown in Table 1 . Changes in relative expression of different samples were calculated by the ABI Q6 Fast real-time PCR system (Applied Biosystems, Foster City, CA, USA) using the 2−∆∆CT method. GAPDH was used as an internal reference gene.
Primer sequences used for qRT-PCR analyses
| Gene | Primer | Sequence |
|---|---|---|
| Mouse GAPDH | m-GAPDH-F | CCAGAGCTGAACGGGAAGCTCAC |
| m-GAPDH-R | CCATGTAGGCCATGAGGTCCACC | |
| Mouse CXCL1 | m-CXCL1-F | AGCTGCGCTGTCAGTGCC |
| mCXCL1-R | CAAGCCTCGCGACCATTC | |
| Mouse IL-6 | m-IL6-F | CTCCCAACAGACCTGTCTATAC |
| m-IL6-R | CCATTGCACAACTCTTTTCTCA | |
| Mouse IL-1β | m-IL1β-F | AAATGCCACCTTTTGACAGTGA |
| m-IL1β-R | GGTTTGGAAGCAGCCCTTCA | |
| Mouse TNF-α | m-TNFα-F | GATCGGTCCCCAAAGGGATG |
| m-TNFα-R | CCACTTGGTGGTTTGTGAGTG | |
| Mouse P300 | m-P300-F | GCCAACATTGGAGGCACTTT |
| m-P300-R | GCCAACATTGGAGGCACTTT |
5
Quantitative Analysis of HSV-1 Viral Genes
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Nucleospin RNA Kit (Takara, Dalian, China) was used to extract the total RNA from samples. cDNA was synthesized using Primescript RT Master Mix (Transgen, Beijing, China) obeying the manufacturer’s guidance. qPCR was launched using SYBR Premix Ex Taq II (Vazyme, Nanjing, China) and Applied Biosystems 7500 Real-Time PCR system (Applied Biosystems, Foster City, USA) to measure HSV-1 virus DNA. The cycle condition was 95°C for 30s, followed by 40 two-step cycles (95°C for 5s, 60°C for 30s). The primers for the genes RPL5, HSV-1 ICP0, TK, VP16, mrpl5, il-6, il-1α, tnf-α, nlrp3, caspase 1 and il-1β are provided in Table 1 and the relative gene expression was determined based on deactivation with the housekeeping gene as described previously.30 (link)
Primers Used for qPCR
| No. | Gene | Forward Primer | Reverse Primer |
|---|---|---|---|
| 1. | RPL5 | GGAAGCACATCATGGGTCAGA | TACGCATCTTCATCTTCCTCCATT |
| 2. | ICP0 | GGCCCCCTTGTCAACAGA | GGGAGTCGCTGATCACTATGG |
| 3. | VP16 | GCGGGGCCGGGATTTACC | CTCGAAGTCGGCCATATCCA |
| 4. | TK | AAGGTCGGCGGGATGAG | CGGCCGCGCGATAC |
| 5. | il1b | CTTTCCCGTGGACCTTCCA | CTCGGAGCCTGTAGTGCAGTT |
| 6. | tnf-a | ACAAGGCTGCCCCGACTAC | TGGGCTCATACCAGGGTTTG |
| 7. | il6 | ACCACTCCCAACAGACCTGTCT | CAGATTGTTTTCTGCAAGTGCAT |
| 8. | il1a | GATGAAGCTCGTCAGGCAGAA | CCTCCCGACGAGTAGGCATA |
| 9. | casp1 | CTGGGACCCTCAAGTTTTGC | CCCTCGGAGAAAGATGTTGAAA |
| 10. | m-rpl5 | CCGCAGGCTTCTGAATAGGT | CCAGTTGTAGTTCGGGCAAGA |
| 11. | nlrp3 | CTGCGGACTGTCCCATCAAT | AGGTTGCAGAGCAGGTGCTT |
6
Quantitative RT-PCR for Gene Expression
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Bacterial overnight cultures were inoculated into fresh LB medium and grown to an OD600 of 1.0. Total RNA was extracted and reverse transcribed to cDNA. The cDNA was mixed with the indicated primers (Table S4 in Supplemental File 1) and SYBR premix Ex Taq II (Vazyme, Nanjing, China). Then, qRT–PCR was performed in a Step One Plus system (Life Technologies, USA). The DNA-directed RNA polymerase beta chain gene rpoB was used as an internal control.
7
Quantitative Real-Time PCR Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
After treated with DMSO or BD for 48 h, the cells were collected. Total RNA was extracted from cells using TRIzol™ reagent (Invitrogen) according to the manufacturer’s instructions. quantity real-time PCR (qRT-PCR) was conducted as previous report (Dong et al., 2020 (link)). Briefly, the total RNA was reversely transcribed to cDNA by using the M-MLV reverse transcriptase (Promega). SYBR PreMix Ex Taq II (Vazyme Biotech, Nanjing, China) was used to carry out qRT-PCR in 20 μL reaction system. RT-qPCR reaction was carried out by using a LightCycle96 real-time PCR system (Roche). The relative mRNA expression level was calculated by 2−ΔΔCT method. The primers used in this study are listed in Table 1 .
8
Quantification of PTGS2 and HMOX1 mRNA in Osteosarcoma
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total RNA was extracted from osteosarcoma cells using Trizol Reagent (Invitrogen, California, United States) according to the manufacturer’s instruction. First strand cDNA synthesis and amplification were performed using PrimeScript™ RT reagent Kit with gDNA Eraser (TAKARA, Shiga, Japan). q-PCR was used to quantify the expression of PTGS2 and HMOX1 mRNAs using SYBR® Premix Ex TaqII (Vazyme, Nanjing, China). GAPDH was used as the internal control gene. Gene specific primers were purchased from Genewiz Biological Technology (Suzhou, China). The primer sequences for q-RCR were listed as below: PTGS2, forward 5′-TGTGCAACACTTGAGTGGCT-3′, reverse 5′-ACTTTCTGTACTGCGGGTG-3’. HMOX1, forward 5′-GAAGAAGGTGCTAATGTCCTGAC-3′, reverse 5′-GTCCCAGACTGTAATTTCGCC-3’. GAPDH, forward 5′-AGATCCCTCCAAAATCAAGTGG-3′, reverse 5′-GGCAGAGATGATGACCCTTTT-3’.
9
Quantitative Analysis of RNA Expression
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total RNA was extracted by using an RNAiso Plus reagent (AL11817A, TaKaRa, Beijing, China) according to protocol. The total RNA extracted from CHIR- and sh-AXIN2CHIR- piPSCs were collected after the CHIR99021 withdrawal for three passages, and the total RNA extracted from sh-AXIN1, sh-AXIN2, OE-AXIN1, and OE-AXIN2 ipPSCs were collected after cell line purification. Then, by using the PrimeSript™ RT reagent Kit (Tiangen, China) according to its protocol, the cDNA was synthesized as a reverse transcription PCR (RT-PCR). The qRT-PCR reaction system was 20 μL in volume: 10 μL SYBR® Premix Ex Taq II (Vazyme), 0.5 μL cDNA, 0.4 μL PCR Forward Primer (10 μM), 0.4 μL PCR Reverse Primer (10 μM), and RNase-free water added to a total volume of 20 μL.
10
Inflammatory Pathway Induction and Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
DMEM, RPMI 1640, and Opti-MEM medium were purchased from Thermo Fisher Scientific (Waltham, MA, USA). Fetal bovine serum, Hoechst 33342, propidiumiodide (PI), penicillin G sodium salt, lipopolysaccharide (LPS), Freund’s complete adjuvant, and chicken type II collagen were obtained from Merck (Darmstadt, Germany). Antibodies against NLRP3, GSDMD, caspase-1, NF-κB, and p-NF-κB were obtained from Cell Signaling Technology (Danvers, MA, USA). Antibodies against Tubulin, STING, p-STING, F4/80, and γ-H2AX were obtained From Abcam (Cambridge, United Kingdom). Mouse IL-1β and IL-18 uncoated ELISA kits were purchased from Thermos Fisher Scientific (Waltham, MA, USA). Lactate dehydrogenase (LDH) assay kits were obtained from Nanjing Jiancheng Bioengineering Institute (Nanjing, China). TRIzol reagent, PrimeScript RT Reagent Kit with gDNA Eraser, Effectene Transfection Reagent, and SYBR Premix Ex TaqII were obtained from Vazyme (Nanjing, China). CCCP and JSH-23 were purchased from MCE (New Jersey, USA). Human or mouse PBMC separation medium were obtained from Solarbio (Beijing, China).
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!