RNA was extracted from cells using a RNeasy kit (Qiagen). Approximately 2 μg of RNA from each cell line was converted to cDNA using a High-Capacity cDNA Reverse Transcription Kit (Thermo), and subsequently combined with water to make a 1:5 dilution. To measure TERT expression, a 20 μl RT-PCR reaction was prepared with 1x iQ SYBR Green Supermix reagents (Biorad), 3 μl of diluted cDNA, and 500 nM of the primer pair for TERT or TBP (TERT_Ex9-10_forward 5’-AGTGCCAGGGGATCCCGCA, TERT_Ex9-10_reverse 5’-GAGGTGTCACCAACAAGAAATCATCC, TBP_forward 5’-CACGAACCACGGCACTGATT, TBP_reverse 5’-TTTTCTTGCTGCCAGTCTGGAC). The following cycling conditions were used: 1 cycle of 95 °C for 3 min, 35 cycles of 95 °C for 25 s and 60 °C for 30 s. The expression of TERT was normalized to TBP expression (2-ΔCt).
Iq sybr green supermix reagent
Manufactured by Bio-Rad
Sourced in United States, Canada
IQ SYBR green supermix reagent is a complete, ready-to-use solution for real-time PCR amplification. It contains SYBR Green I dye, which binds to double-stranded DNA and emits fluorescence. The reagent also includes a DNA polymerase, dNTPs, and necessary buffers and salts for efficient DNA amplification.
Automatically generated - may contain errors
Lab products found in correlation
Trizol reagent, by Thermo Fisher Scientific (27 mentions)
Real time pcr machine, by Bio-Rad (15 mentions)
Iscript cdna synthesis kit, by Bio-Rad (9 mentions)
Rneasy kit, by Qiagen (5 mentions)
Nanodrop spectrometer, by Thermo Fisher Scientific (4 mentions)
Iscript reverse transcription supermix, by Bio-Rad (4 mentions)
Direct zol rna miniprep kit, by Zymo Research (4 mentions)
Option 2, by Bio-Rad (3 mentions)
Cfx96 real time pcr system, by Bio-Rad (3 mentions)
Nucleospin rna kit, by Macherey-Nagel (3 mentions)
Icycler real time pcr machine, by Bio-Rad (3 mentions)
Primescript rt reagent kit, by Takara Bio (3 mentions)
Mirna specific primer, by RiboBio (2 mentions)
Iscript kit, by Bio-Rad (2 mentions)
High capacity cdna reverse transcription kit, by Thermo Fisher Scientific (2 mentions)
Abi 7500 real time pcr system, by Thermo Fisher Scientific (2 mentions)
Rneasy mini kit, by Qiagen (2 mentions)
Cfx connect instrument, by Bio-Rad (2 mentions)
Opticon 2 real time pcr machine, by Bio-Rad (2 mentions)
Direct zol rna miniprep, by Zymo Research (2 mentions)
74 protocols using iq sybr green supermix reagent
1
TERT Expression Quantification by RT-PCR
2
Adrenal Gland Zone-Specific RNA Analysis
3
Real-time PCR for Gene Expression Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Real-time PCR were performed on an ABI 7500 Real-time PCR System (Applied Biosystems) using iQ SYBR Green Supermix reagents (BIO-RAD) according to the Manufacturer’s specification. The reaction was performed in a 20 μL system containing 10 μL 2 × iQ SYBR Green Supermix, 0.5 μL of 20 μM forward primer, 0.5 μL of 20 μM reverse primer, 0.8 μL of cDNA and 8.2 μL double-distilled water (ddH2O). The real-time PCR was carried out as follows: 1 cycle of 95°C for 5 min, followed by 40 cycles of 95°C for 15 s and 60°C for 35 s. Fluorescent signals were collected during the elongation step.
4
Quantitative Analysis of miRNA and CircRNA
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For miR-342-3p, RNA was reverse-transcribed using miRNA-specific primer (Ribobio, Guangzhou, China). For circ_0006220, TADA2A, and GOT2, complementary DNA (cDNA) was synthesized using commercial Bio-Rad iScript kit (Bio-Rad, Hercules, CA, USA). qPCR reaction was implemented via commercial iQ SYBR Green Supermix reagents (Bio-Rad) and primers (Supplementary Table 1
5
Quantitative Analysis of MET Gene Expression
6
Evaluating Wound Healing Modulators
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The siRNA oligonucleotides were purchased from either Qiagen (Valencia, CA) or Dharmacon (Boulder, CO). Histidine-lysine co-polymer (HKP) was provided by Biopolymer lab at the University of Maryland or purchased from Ambio Pharma Inc. (Shanghai, China). Primers PCR were purchased from Qiagen (Germantown, MD) or Genepharm (Suzhou, China). Rabbit polyclonal anti-alpha smooth muscle Actin (α-SMA) antibody, Rabbit polyclonal anti-COX2 antibody, Rabbit monoclonal anti-TGF-β1, Rabbit Monoclonal anti-human MHC class 1 antibody, Rabbit polyclonal antibody to CD31, and Rabbit polyclonal antibody to VEGF were purchased from Abcam Corporation (Iowa, USA). Rabbit monoclonal anti-SMAD2/3, anti-p-SMAD2/3 and anti PAI-1 antibody were purchased from Cell Signaling Technology (Danvers, MA), The Bio-Rad iScript reverse transcription kit and IQ Sybr Green Supermix reagents (Hercules, CA) were used for qRT-PCR performed with Bio-Rad MyiQ Thermal Cycler. COX2 inhibitor Celecoxib (Catalog No.S1261) and TGFβ receptor I (TBR1) antagonist Galunisertib (LY2157299) (Catalog No.S2230) were purchased from Selleck Chemicals (Shanghai, China).
7
Quantitative Analysis of RNA Transcripts
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Trizol was used to extract total RNA in accordance with the manufacturer's instructions. For miR‐1299, RNAs were reverse‐transcribed using a miRNA‐specific primer (Ribobio). For hsa_circ_0001925, kinesin family member 4A (KIF4A) and YY1, complementary DNA (cDNA) was synthesized using a commercial Bio‐Rad iScript kit (Bio‐Rad). qPCR reaction was enforced via commercial iQSYBR Green Super‐Mix reagents (Bio‐Rad) and primers (Table 1 ). The relative abundance of molecules was analyzed according to the 2−ΔΔCt method.17
8
Quantitative RT-PCR Analysis of Kidney Gene Expression
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total RNA was extracted from the kidney tissues with TRIzol reagent (Invitrogen, Carlsbad, CA). Aliquots (1 μg) of total RNA were reverse transcribed using SuperScript II reverse transcriptase. Quantitative Real-Time PCR was performed using IQ SYBR green supermix reagent (Bio-Rad, Herculus, CA) with a Bio-Rad real-time PCR machine according to the manufacturer's instructions. The comparative Ct method (ΔΔCt) was used to quantify gene expression, and the relative quantification was calculated as 2−ΔΔCt. [12 (link), 22 (link), 36 (link)] The expression levels of the target genes were normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) level in each sample. The primer sequences were as follows: CXCL16 - forward 5′-ACCCTTGTCTCTTGCGTTCTTCCT-3′, reverse 5′-ATGTGATCCAAAGTACCCTGCGGT-3′; IL-6 - forward, 5′-AGGATACCACTCCCAACAGACCTG-3′, reverse, 5′-CTGCAAGTGCATCATCGTTGTTCA-3′; TNFα - forward, 5′-CATGAGCACAGAAAGCATGATCCG-3′, reverse, 5′-AAGCAGGAATGAGAAGAGGCTGAG-3′; IL-1β - forward, 5′-CTTCAGGCAGGCAGTATCACTCAT-3′, reverse, 5′-TCTAATGGGAACGTCACACACCAG-3′; TGF-β1 - forward, 5′-CAACAATTCCTGGCGTTACCTTGG-3′, reverse, 5′-GAAAGCCCTGTATTCCGTCTCCTT-3′; and GAPDH - forward, 5′-CCAATGTGTCCGTCGCGTGGATCT-3′, reverse, 5′-GTTGAAGTCGCAGGAGACAACC-3′.
9
Thapsigargin-Induced ER Stress in N2a Cells
10
Quantitative Real-Time PCR Analysis of Renal Gene Expression
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total RNA was extracted from the renal cortical tissues and cultured renal cell lines. Real-time PCR was carried out with machine (Option 2, Bio-Rad, Hercules, CA, USA) by using IQ SYBR Green Supermix reagent (Bio-Rad) 16 (link), 17 . The Sequence of RNA Primers used such as Smad3, Smad7, collagen-I, α-SMA, MCP-1, NF-kB, ERBB4-IR, LRNA9884, and GAPDH were described previously 5 (link), 7 (link), 12 (link), 18 (link). The house keeping genes β-actin was used as internal controls. The ratio of specific mRNA to β-actin mRNA was calculated using the 2-ΔCt method and is expressed as the mean ± S.E.M.
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!