Total RNA was isolated from cells or clinical tumor samples with Ultrapure RNA kit (CWBiotech) and the complementary DNA (cDNA) was synthesized by using Transcriptor First Strand cDNA Synthesis System kit (Roche) with Oligo (dT)18. For semi-quantitative analysis, PCR amplification was performed with GoTaq® DNA polymerase (Promega), 1 μM of each pair of the indicated primers (Additional file 1 : Table S1), and 0.5 μl cDNA for a 2 min initial denaturation at 95 °C, followed by 20–27 cycles of 30 s at 95 °C, 30 s at the appropriate anneal time, and 1 min at 72 °C. Products were run in 1.5% agarose gel and the band intensity was scanned, and normalized based on their corresponding internal GAPDH control as the relative expression level. Real time PCR was performed by using a SYBR Green reaction kit (TIANGEN, China) in CFX 96 real time PCR instrument(BioRad,USA), according to the supplier’s protocol. All experiments were carried out in triplicate and analyzed using the comparative threshold cycle (2−ΔΔCT) method, where the ΔΔCT is the difference between normalized target gene and internal control (ΔCT sample – ΔCT control = ΔΔCT).
Sybr green reaction kit
Manufactured by Tiangen Biotech
Sourced in China
The SYBR Green reaction kit is a laboratory product designed for real-time polymerase chain reaction (PCR) experiments. It contains the necessary reagents, including a SYBR Green dye, to detect and quantify DNA amplification during the PCR process. The kit provides a simple and efficient way to perform real-time PCR analysis.
Automatically generated - may contain errors
Lab products found in correlation
96 well plate, by Promega (2 mentions)
In nite m200, by Tecan (2 mentions)
Transcriptor first strand cdna synthesis system kit, by Tiangen Biotech (2 mentions)
Trizol reagent, by Takara Bio (2 mentions)
Cfx96 real time pcr instrument, by Bio-Rad (1 mentions)
Gotaq dna polymerase, by Promega (1 mentions)
Ultrapure rna kit, by CWBIO (1 mentions)
3 protocols using sybr green reaction kit
1
Semi-Quantitative and Real-Time PCR Analysis
2
Cell Viability Assay for PCBP1 Overexpression
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Cell viability was investigated using the methyl tetrazolium salt (MTS) assay. Cells were plated into 96-well plates (Promega, Beijing, China) and incubated 24 h. Then used transfection reagent to transfect pEGFP-N1 or pEGFP-N1-PCBP1 and cultured 24 h and 48 h to detect viability. 20 µl / well of MTS solution was added to each 100 µl of DMEM medium, and incubated for 60 min at a 37℃ constant temperature incubator. Then the absorbance was detected with multifunction microplate reader (Tecan In nite M200, Swiss) at 490 nm. The survival rate of cells in each well was shown as a percentage of control.
Quantitative RT-PCR analysis and agarose gel electrophoresis Total RNA was extracted from cells with TRIzol reagent (Takara Biotech Co., Ltd.) and the complementary DNA (cDNA) was synthesized by using Transcriptor First Strand cDNA Synthesis System kit, real-time PCR analysis of PCBP1 and the reference gene β-Actin was treated by using a SYBR Green reaction kit (TIANGEN, China) in real-time PCR instrument (Thermo, USA), according to instruction. All experiments were carried out in triplicate and analyzed using the comparative threshold cycle (2 -ΔΔCT ) method. Products were run in 1% agarose gel and the band intensity was scanned. The results were normalized by β-Actin levels.
Quantitative RT-PCR analysis and agarose gel electrophoresis Total RNA was extracted from cells with TRIzol reagent (Takara Biotech Co., Ltd.) and the complementary DNA (cDNA) was synthesized by using Transcriptor First Strand cDNA Synthesis System kit, real-time PCR analysis of PCBP1 and the reference gene β-Actin was treated by using a SYBR Green reaction kit (TIANGEN, China) in real-time PCR instrument (Thermo, USA), according to instruction. All experiments were carried out in triplicate and analyzed using the comparative threshold cycle (2 -ΔΔCT ) method. Products were run in 1% agarose gel and the band intensity was scanned. The results were normalized by β-Actin levels.
3
Cell Viability Assay for PCBP1 Overexpression
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cell viability was investigated using the methyl tetrazolium salt (MTS) assay. Cells were plated into 96-well plates (Promega, Beijing, China) and incubated 24 h. Then used transfection reagent to transfect pEGFP-N1 or pEGFP-N1-PCBP1 and cultured 24 h and 48 h to detect viability. 20 µl / well of MTS solution was added to each 100 µl of DMEM medium, and incubated for 60 min at a 37℃ constant temperature incubator. Then the absorbance was detected with multifunction microplate reader (Tecan In nite M200, Swiss) at 490 nm. The survival rate of cells in each well was shown as a percentage of control.
Quantitative RT-PCR analysis and agarose gel electrophoresis Total RNA was extracted from cells with TRIzol reagent (Takara Biotech Co., Ltd.) and the complementary DNA (cDNA) was synthesized by using Transcriptor First Strand cDNA Synthesis System kit, real-time PCR analysis of PCBP1 and the reference gene β-Actin was treated by using a SYBR Green reaction kit (TIANGEN, China) in real-time PCR instrument (Thermo, USA), according to instruction. All experiments were carried out in triplicate and analyzed using the comparative threshold cycle (2 -ΔΔCT ) method. Products were run in 1% agarose gel and the band intensity was scanned. The results were normalized by β-Actin levels.
Quantitative RT-PCR analysis and agarose gel electrophoresis Total RNA was extracted from cells with TRIzol reagent (Takara Biotech Co., Ltd.) and the complementary DNA (cDNA) was synthesized by using Transcriptor First Strand cDNA Synthesis System kit, real-time PCR analysis of PCBP1 and the reference gene β-Actin was treated by using a SYBR Green reaction kit (TIANGEN, China) in real-time PCR instrument (Thermo, USA), according to instruction. All experiments were carried out in triplicate and analyzed using the comparative threshold cycle (2 -ΔΔCT ) method. Products were run in 1% agarose gel and the band intensity was scanned. The results were normalized by β-Actin levels.
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